Ariel Gore

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.

Ocular Surface Changes After Sulfur Mustard Exposure in Rabbits, Monitored by Impression Cytology

Purpose: Ocular injuries after exposure to sulfur mustard (SM) are characterized by acute corneal erosion and inflammation of the anterior segment that may be followed by delayed corneal neovascularization and epithelial defects, associated with limbal stem cell deficiency in part of the exposed eyes. This study aimed to further clarify the mechanism of the late injury by monitoring SM-induced cytological alterations in the ocular surface, in relation to the clinical symptoms, using impression cytology (IC). Methods: Rabbit eyes were exposed to SM vapor (n = 20) and were clinically observed up to 4 weeks. Samples for IC were collected simultaneously from the upper bulbar conjunctiva, limbus, and cornea and then fixed and stained with periodic acid–Schiff and hematoxylin. At 1 month, animals were killed and eyes dissected and processed for histology. Results: Concomitant with clinical symptoms of SM ocular toxicity, IC showed significant long-term loss of conjunctival goblet cells shortly after exposure, followed by abnormal differentiation toward squamous metaplasia. Simultaneously with corneal erosion, apoptotic bodies and cellular debris were seen in the corneal epithelium, followed by regeneration at 1 week. Migration of conjunctival goblet cells toward the cornea was noted in neovascularized eyes, as early as 1 week, indicating limbal stem cell deficiency. The IC findings were supported by histological evaluation. Conclusions: Continuous monitoring of the ocular surface after SM exposure by IC enables earlier detection of pathology and therapeutic intervention, therefore, is recommended for routine follow-up of casualties. Prolonged loss of goblet cells may point toward the role of mucin in the pathogenesis.

Successful single treatment with ziv-aflibercept for existing corneal neovascularization following ocular chemical insult in the rabbit model

Purpose To evaluate the efficacy of ziv‐aflibercept as a treatment for established corneal neovascularization (NV) and to compare its efficacy to that of bevacizumab following ocular chemical insult of sulfur mustard (SM) in the rabbit model. Methods Chemical SM burn was induced in the right eye of NZW rabbits by vapor exposure. Ziv‐aflibercept (2 mg) was applied once to neovascularized eyes by subconjunctival injection while subconjunctival bevacizumab (5 mg) was administered twice a week, for 3 weeks. Non‐treated exposed eyes served as a control. A clinical follow‐up employed by slit‐lamp microscope, was performed up to 12 weeks following exposure and digital photographs of the cornea were taken for measurement of blood vessels length using the image analysis software. Eyes were taken for histological evaluation 2, 4 and 8 weeks following treatment for general morphology and for visualization of NV, using H&E and Masson Trichrome stainings, while conjunctival goblet cell density was determined by PAS staining. Results Corneal NV developed, starting as early as two weeks after exposure. A single subconjunctival treatment of ziv‐aflibercept at 4 weeks post exposure, significantly reduced the extent of existing NV already one week following injection, an effect which lasted for at least 8 weeks following treatment, while NV in the non‐treated exposed eyes continued to advance. The extensive reduction in corneal NV in the ziv‐aflibercept treated group was confirmed by histological evaluation. Bevacizumab multiple treatment showed a benefit in NV reduction, but to a lesser extent compared to the ziv‐aflibercept treatment. Finally, ziv‐aflibercept increased the density of conjunctival goblet cells as compared to the exposed non‐treated group. Conclusions Subconjunctival ziv‐aflibercept single treatment presented a highly efficient long‐term therapeutic benefit in reducing existing corneal NV, following ocular sulfur mustard exposure. These findings show the robust anti‐angiogenic efficacy of ziv‐aflibercept and demonstrate the advantage of this treatment over the other anti‐angiogenic therapies in ameliorating corneal NV and protecting the ocular surface. HighlightsA single subconjunctival 2 mg ziv‐aflibercept (80 &mgr;l) treatment drastically reduced existing corneal NV already one week following treatment.The ziv‐aflibercept treatment presented a prolonged and higher efficacy in ameliorating corneal NV than the bevacizumab treatment.The ziv‐aflibercept treatment dramatically improved the conjunctival goblet cell density.

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.

Differential expression of corneal and limbal cytokines and chemokines throughout the clinical course of sulfur mustard induced ocular injury in the rabbit model

Purpose: The sight threatening sulfur mustard (SM) induced ocular injury presents specific symptoms for each clinical stage. The acute injury develops in all of the exposed eyes and is characterized by erosions and severe inflammation. The irreversible late pathology develops only in part of the eyes, and is clinically expressed by chronic inflammation and corneal neovascularization (NV). The mechanisms underlying this injury are still in research and treatment is insufficient. Aiming to shed light on pathological mechanisms and improve the therapeutic measures, we studied the expression pattern of various cytokines and chemokines at different clinical stages of the ocular injury. Methods: Rabbit right eye was exposed to SM vapor and a clinical follow‐up was carried out up to 4 weeks. Corneal and limbal tissues were collected at 48 h, 1w and 4w post exposure and IL‐1&agr;, IL‐1&bgr;, IL‐6, TNF&agr;, macrophage chemotactic protein (MCP)‐1 and IL‐8 levels were measured by commercial ELISA kits. Results: SM exposed eyes presented an acute injury that was partially resolved within a week in all of the exposed eyes, and was followed by an irreversible late pathology in 50%–80% of the eyes, beginning at 2w. A significant elevation was seen in levels of the studied factors, however each factor presented a unique expression pattern. At the peak of the acute injury, at 48 h, significantly higher levels of corneal IL‐1&agr;, IL‐8, and TNF&agr; and limbal IL‐1&agr; and MCP‐1 were found compared to naïve eyes. At 1w, corneal IL‐1&bgr;, IL‐6, IL‐8 and TNF&agr; and limbal IL‐8 and MCP‐1 levels were significantly higher compared to naïve eyes. During the late pathology, at 4w, elevated levels of corneal IL‐1&bgr;, IL‐6 and MCP‐1 and limbal MCP‐1 and IL‐8 were found only in eyes presenting NV. Conclusions: The levels of the studied factors changed throughout the dynamic course of the ocular injury. The prolonged increased levels of limbal MCP‐1 and IL‐8 may contribute to the continuous recruitment of inflammatory cells, characterizing the symptoms of the late pathology. The significantly elevated IL‐1&bgr; and IL‐6 at 1w, after the resolution of the acute injury but before the clinical manifestation of the late pathology suggests a therapeutic window for intervention with prevention therapy. Mapping the expression pattern of these cytokines and chemokines points out towards stage‐specific therapeutic options.