J. Turetz

Ocular injuries following sulfur mustard exposure: Pathological mechanism and potential therapy

Sulfur mustard (SM) is a potent vesicant, known for its ability to cause incapacitation and prolonged injuries to the eyes, skin and respiratory system. The toxic ocular events following sulfur mustard exposure are characterized by several stages: photophobia starting a few hours after exposure, an acute injury phase characterized by inflammation of the anterior segment and corneal erosions and a delayed phase appearing following a clinically silent period (years in human). The late injury appeared in part of the exposed eyes, expressed by epithelial defects and corneal neovascularization (NV), that lead to vision deficits and even blindness. During the last years we have characterized the temporal development of ocular lesions following SM vapor exposure in rabbits and have shown the existence of two sub-populations of corneas, those exhibiting delayed ocular lesions (clinically impaired) and those exhibiting only minor injuries if at all (clinically non-impaired). The aim of the present study was to investigate the pathological mechanism underlying the delayed injury by focusing on the unique characteristics of each sub-population and to test the efficacy of potential treatments. Clinically impaired corneas were characterized by chronic inflammation, increased matrix metalloproteinase (MMP) activity, poor innervation and limbal damage. Moreover, using impression cytology and histology, we identified the delayed lesions as typical for an ocular surface disorder under the category of limbal epithelial stem cell deficiency (LSCD). These results point to therapeutic directions, using anti-inflammatory drugs, MMPs inhibitors, neurotrophic factors and amniotic membrane transplantation. Topical anti-inflammatory drugs, either steroid (Dexamycin, DEX) or non-steroidal anti-infllammatory drug (NSAID, Voltaren Ophtha) were found to be beneficial in ameliorating the initial inflammatory response and in postponing the development of corneal NV, when given during the first week after exposure. When DEX was administered as a symptomatic treatment against NV, a significant regression in the angiogenic process was observed, however, the effect was temporal and blood vessels reappeared after therapy ceased. Chronic administration (8 weeks) of the MMP inhibitor Doxycycline was also effective in attenuation of the acute and delayed injury. Preliminary results, using amniotic membrane transplantation revealed some decrease of corneal edema with no effect on corneal NV. It is suggested that the chronic inflammation and prolonged impairment of corneal innervation are playing a role in the pathogenesis of the delayed LSCD following SM exposure by creating a pathological microenvironment to limbal epithelial stem cells, thus, leading to their slow death and to a second cascade of pathological events eventually resulting in severe long-term injuries. As of today, only topical anti-inflammatory drugs reached the criteria of an applicable efficient post-exposure ocular treatment for SM injuries. Further studies are required to investigate the effects of SM on epithelial stem cells and their involvement in the pathogenesis of the long-term injuries.

Characterization of acute and delayed ocular lesions induced by sulfur mustard in rabbits

Purpose. To establish an experimental model for sulfur mustard-induced acute and delayed ocular lesions in rabbits. Methods. Rabbit eyes were exposed to sulfur mustard (HD) vapor (370, 420 µg/l) for a period of two minutes. A three months follow-up study was carried out, based on the evaluation of clinical, biochemical and histological parameters. Results. HD exposure initiated typical clinical symptoms within 2–6 hrs, characterized by eye closure, eyelid swelling, conjunctival hyperemia, corneal erosions and inflammation. The clinical signs were significantly dose-dependent and reached a peak at 24–72 hrs post exposure. Biochemical evaluation of the aqueous humor exhibited an inflammatory reaction and oxidative stress at 4 hrs after exposure, subsiding at 28 hrs after exposure. Histological examination of corneas at 48 hrs revealed epithelial denudation and marked stromal edema, accompanied by cellular infiltration. Epithelial regeneration started after 72 hrs, and recovery was almost completed within 1Ð2 weeks, depending on the HD dose. A second phase of pathological processes started as early as two weeks post exposure and was characterized by corneal edema, opacity, recurrent erosions and neovascularization. The delayed injuries were found in 25 and 40% of the eyes respectively, and when appearing, were more severe than the initial ones. Conclusions. The development of HD-induced ocular lesions in rabbits is similar to the lesions described in human casualties. Quantitative analysis of the various clinical parameters emphasizes the contribution of each tissue to the overall toxic picture. Our experimental model is useful for studying the pathological mechanisms of HD-ocular lesions, and may serve for testing potential therapies.

The Beneficial Effects of Doxycycline, An Inhibitor of Matrix Metalloproteinases, on Sulfur Mustard-Induced Ocular Pathologies Depend on the Injury Stage

Abstract Purpose: Sulfur mustard (SM) induces acute ocular lesions, including erosions and inflammation that may be followed by delayed injuries expressed by epithelial defects and neovascularization (NV). Based on the matrix metalloproteinases (MMPs) activity, we evaluated the clinical and biochemical effects of topical treatment with doxycycline, an MMP inhibitor, targeted to the various injury stages. Methods: Rabbit eyes were exposed to SM vapor. A clinical follow-up was carried out up to 2 months. Tear fluid and cornea samples were collected at different time points for measurements of MMPs activity by zymography. Efficacy of a post-exposure topical doxycycline (2 mg/ml in phosphate buffer saline, ×4/d), targeted to the different phases of the clinical injury, was evaluated. Results: Elevated MMP-9 and MMP-2 activities were found in all corneas during the acute injury and in vascularized corneas during the delayed pathology. In the tear fluid, high MMP-9 activity and negligible MMP-2 activity were found in all the exposed eyes until after the appearance of the delayed pathology symptoms. Prolonged doxycycline treatment reduced MMP-9 activity in the tear fluid. During the acute phase, doxycycline treatment reduced corneal MMP-9 activity and the severity of the injury. Targeting the delayed pathology, doxycycline was clinically efficient only when treatment began before NV appearance. Conclusions: This in vivo study showed the involvement of MMP-9 and MMP-2 during different phases of the SM-induced ocular injury, and the potential of doxycycline treatment as a post exposure measure for reducing the acute injury and as a preventive therapy for ameliorating the delayed pathology. The tear fluid provided a non-invasive method for continuous follow-up of MMPs activity and revealed additional beneficial aspects of injury and the treatment.

Cultivation and characterization of limbal epithelial stem cells on contact lenses with a feeder layer: toward the treatment of limbal stem cell deficiency.

Purpose: Limbal epithelial sheets are used to promote corneal surface reconstruction after the detection of limbal epithelial stem cell deficiency. The aim of this study was to evaluate a novel combination of limbal stem cells (LSCs) maintained on contact lenses (CLs) in the presence of a 3T3 feeder cell layer regarding preservation of stem cell phenotype and the potential use for future in vivo transplantation. Methods: Limbal epithelial cells were isolated from rabbit cornea and cultured with 3T3 cells on CLs. The preservation of LSC phenotype was determined using p63&agr; and ABCG2 immunostaining, whereas epithelial differentiation was evaluated using CK3 and CK19. The colony-forming assay was used to determine the percentage of LSCs in cultures. Finally, CLs seeded with PKH26-labeled LSCs were transferred to rabbit eyes after performing a surgical keratectomy, and the transition and phenotype of labeled cells on the corneal surface were evaluated in whole-mount corneas. Results: Proliferation of individual limbal cells was observed on CLs with a 3T3 feeder cell layer, showing holoclone formation and retention of viable stem or progenitor cell phenotype. Finally, a higher transition of cultivated cells after a dual sequential CL transplantation to the ocular surface was observed, showing the preservation of the LSC phenotype in the corneal surface. Conclusions: Limbal cells cultivated on a CL carrier overlaying a 3T3 feeder layer are mitotically active and retain the LSC phenotype. This novel technique of using CLs as a carrier offers an easily manipulable and nonimmunogenic method for transferring LSCs for ocular surface reconstruction in patients with limbal epithelial stem cell deficiency.

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.

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.