Eliezer Fishbine

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.

Delayed Loss of Corneal Epithelial Stem Cells in a Chemical Injury Model Associated with Limbal Stem Cell Deficiency in Rabbits

Purpose: Ocular injuries following exposure to the chemical agent sulfur mustard (SM) are characterized by acute corneal erosions and inflammation of the anterior segment that may be followed by delayed Partial Limbal Stem Cell Deficiency (LSCD), expressed clinically by corneal neovascularization and epithelial defects. LSCD may derive from direct destruction of limbal stem cells or indirectly from altered limbal stromal niche. The aim of this study was to investigate the mechanism underlying LSCD in SM injuries, focusing on the effects of the chemical on limbal epithelium. Methods: Rabbit eyes were exposed to SM vapor and were observed by slit lamp examinations and pachymetry. Eyes were taken for histological and molecular biology evaluations at different time points (4 h–4 weeks), to include acute and delayed injuries. Epithelial stem cells were identified by ABCG2, p63 and by in vivo BrdU labeling for slow cycling cells. Results: Limbal stem cells were not damaged during the acute phase following SM exposure, in contrast to the severe injury of the central corneal epithelium. On the contrary, limbal epithelium became activated, responding to corneal insult with a wound healing process, as shown by histology and by transient elevation of the stem cells markers. Simultaneously, inflammation was taking place in the limbal stroma lasting for weeks. A gradual loss of stem cells was observed later-on (2–4 weeks), associated with typical symptoms of LSCD. Conclusions: LSCD associated with SM ocular toxicity was not derived from a direct cytotoxic effect on the epithelial stem cells, but apparently from pathological events at the limbal stroma, that produced an abnormal microenvironment for the stem cells, triggering their gradual death. The results, and in particular the absence of a primary damage to the epithelial stem cells, indicate the presence of a therapeutic window for intervention to avoid the development of the delayed LSCD.

Endothelial cell damage following sulfur mustard exposure in rabbits and its association with the delayed-onset ocular lesions

Objective: Ocular injuries following exposure to the toxic agent sulfur mustard (SM) are characterized by acute corneal erosions and inflammation of the anterior segment that may be followed by delayed corneal injuries, expressed clinically by neovascularization and epithelial defects. The present study aimed to investigate the effects of SM on corneal endothelium (CE) during the acute and delayed phase in relation to the development of the long-term pathology. Methods: Rabbit eyes were exposed to SM vapor. A clinical follow-up including pachymetry for measurement of corneal thickness were conducted up to 3 months following exposure. In vivo analysis of corneal endothelium in the central and peripheral cornea was carried out, using a contact specular microscopy. orphometric analysis of cell area and number of cells was performed, to include the acute and delayed phases. Eyes were taken for histology at different time points following exposure (1 h to 3 months). TUNEL staining (Terminal deoxynucleotidyl transferase dUTP nick end labeling) was conducted for detection of apoptosis during the acute phase. Results: SM induced acute corneal erosions and prolonged anterior segment inflammation. Corneal thickness increased within hours, declined after few days but remained higher compared to baseline value for months after the exposure, indicating a chronic edema. Apoptotic alterations were first observed at 6 h resulting in a significant decline in the number of endothelial cells at 24–48 h following exposure. Healing of the endothelium was relatively fast and at one week the Descemet’s membrane was resurfaced, yet, the density and morphology of the cells was often abnormal. Moreover, histological evaluation revealed deformation and enlargement of many cells (polymegathism and pleomorphism), thickening and double layered Descemet’s membrane. These changes were more pronounced in corneas displaying delayed pathology. Discussion and conclusions: SM induced apoptotic cell death of endothelial cells that was accompanied by corneal edema. The impaired healing of the endothelium, including the decrease in endothelial cell density was associated with the delayed-onset injuries. Since human corneal endothelium is almost amitotic, endothelium toxicity should be taken into consideration when testing potential treatments against ocular injuries following SM exposure.

Characterization of acute and long-term pathologies of superficial and deep dermal sulfur mustard skin lesions in the hairless guinea pig model.

Sulfur mustard induces severe acute and prolonged damage to the skin and only partially effective treatments are available. We have previously validated the use of hairless guinea pigs as an experimental model for skin lesions. The present study aimed to characterize a model of a deep dermal lesion and to compare it with the previously described superficial lesion. Clinical evaluation of the lesions was conducted using reflectance colorimetry, trans‐epidermal water loss and wound area measurements. Prostaglandin E2 content, matrix metalloproteinase‐2 and 9 activity, and histopathology were conducted up to 4 weeks post‐exposure. Sulfur mustard skin injury, including erythema and edema, impairment of skin barrier and wounds developed in a dose‐dependent manner. Prostaglandin E2 content and matrix metalloproteinase‐2 and 9 activities were elevated during the wound development and the healing process. Histological evaluation revealed severe damage to the epidermis and deep dermis and vesications. At 4 weeks postexposure, healing was not completed: significantly impaired stratum corneum, absence of hair follicles, and epidermal hyperplasia were observed. These results confirm the use of the superficial and deep dermal skin injuries in the hairless guinea pigs as suitable models that can be utilized for the investigation of the pathological processes of acute as well as long‐term injuries. These models will be further used to develop treatments to improve the healing process and prevent skin damage and long‐term effects.

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.

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).