EFFECT OF TOPICAL MITOMYCIN-C ON CORNEAL LIMBAL STEM CELLS IN MOUSE MODEL Authors: Asadolah Movahedan M.D. Neda Afshar M.D. and Ali R. Djalilian M.D. *The authors have no financial interest in the subject matter of this e-poster. Corneal Epithelial Biology and Tissue Engineering lab, Department of Ophthalmology and Visual Sciences University of Illinois at Chicago Purpose There have been concerns regarding toxic effects of topical Mitomycin C (MMC), which widely used in ophthalmic procedures, on limbal stem cells of ocular surface. In this study, we aimed to see the possible toxicity of various dosage/durations of topical MMC on corneal limbal stem cells in a mouse model. Introduction Mitomycin-c is an alkylating agent that cross-links adenine and guanine in DNA, thereby blocking DNA synthesis and secondarily inhibiting cell mitosis. MMC shows cytotoxic effects that cannot completely be explained by its DNA cross-linking effect; thus the possible long-term cellular effects of MMC are not clear. Topical MMC is widely used in ophthalmic procedures particularly in trabeculectomy, pterygium and refractive surgery. (1) Limbal stem cell deficiency (LSCD) has been reported with MMC use in trabeculectomy surgery. (2) Likewise, there have been concerns regarding its toxic effects on limbal stem cells in refractive surgery especially with longer duration of application. (2-4) To the best of our knowledge, no other published study has exclusively evaluated, limbal stem cell toxicity of MMC in vivo. Subjects and Methods All the animal experiments were conducted in compliance with the recommendations of the Association for Research in Vision and Ophthalmology. Twelve, 4-6 months old C57/Bl6 (black) mice were used for the experiments. To maximize similarities of the experiment with refractive surgery, the eyes underwent a 2 mm central full thickness corneal epithelial ablation using a blunt scraper, leaving a 1mm rim of corneal epithelial cells while general anesthesia was induced by cocktail of Ketamine and Xylazine. Then the eyes were were exposed to: Topical MMC, 0.01% (group A, n=6) or 0.02% (group B, n=6) (case groups) or The same amount of MMC solvent, Balanced Salt Solution (BSS), (control group, n=12) for variable durations (30 to 90 seconds) applied by 2.5-millimeter filter paper discs, then extensively washed with BSS. Outcome Measures i. ii. Slit-lamp examination was performed on day 1, 4, 7 after the treatment to detect early signs of toxicity in the limbal region: Corneal fluorescein staining Epithelial irregularity And 30 days after the exposure for signs of limbal stem cell deficiency such as: i. ii. i. Neovascularization Epithelial defects Impression cytology of limbal region was performed at 1 month: After sacrificing the animal, the eyes were enucleated, placed on a microscope slide for 2-3 minutes to dry and then rolled over so that the limbal region would touch the slide and leaves the impression of adherent cells on the slide. The slides were examined with Periodic acid Schiff (PAS) staining to detect conjunctival goblet cells. Results- Early toxicity Epithelial irregularity in early examinations after application of BSS, 0.01% or 0.02% MMC for 90 seconds. Percentage of the eyes with minimal epithelial irregularity and FL staining 100% 80% 60% BSS (control) MMC 0.01% 40% MMC 0.02% 20% 0% Day 1 Day 4 Day 7 Time of examination Results- Signs of LSCD Punctate epithelial defects were the most evident finding, no corneal ulcer was developed in control or MMC groups after the initial wound healing. Day 1 (24h-post exposure) Fluorescein Mechanical ablation + Topical 0.02% MMC Mechanical ablation + Topical BSS application Day 30 Fluorescein Day 30 Bright Field Results- Signs of LSCD Corneal neovascularization was measured and quantified according to the percentage of the corneal surface area covered by new vessels and their small branches. Higher dose/durations of MMC (0.02% applied for 90 seconds) [n=6] resulted in significantly more corneal neo-vascularization than BSS [n=6]. (p=0.046) Corneal Neovascularization Average % corneal surface covered by new vessels 100% 80% 60% 40% 20% 62.5% 29.1% 0% BSS MMC (control) 0.02% Results- Impression cytology Average percentage of goblet to epithelial cells per field of microscope Impression cytology of the limbal region, indicating the degree of conjunctivalization, confirmed the presence of goblet cells in all eyes of control and both MMC groups. There was no difference between the eyes treated with 0.01% MMC, 0.02% MMC or BSS in the average percentage of goblet to epithelial cells per field of microscope, compared to each other (p=0.25) and control (p=0.28), (p=0.91) respectively. 10% Impression cytology of limbal region Enucleated globe is rolled over on the slide 8% Arrows : Goblet cells 6% 4% 2% 5.2% 4.5% 5.3% 0% BSS MMC MMC (Control) 0.01% 0.02% BSS MMC 0.02% Discussion Mechanical models of mouse corneal wounding healing is widely used by previous studies. (5) In this study, we used a 2 mm central corneal epithelial ablation leaving a 1mm corneal epithelial cell rim to avoid partial limbal stem cell deficiency. The MMC concentrations used in this study are similar to the concentrations used in refractive surgery; although the duration of application was variable. We could detect early signs of epithelial toxicity in the limbal region. Topical MMC might have similar effects applied during refractive surgeries. These effects of might be evident with longer durations which are typically used for patients with higher risk of fibrosis. The damage to the limbal stem cells will likely remain subclinical given the extensive reserve of limbal stem cells, however, if the corneal regenerative capacity is challenged (eg. subsequent ocular surface damage or surgery) the limbal damage might become manifest. We have designed series of experiments to continue this study in future with evaluation of the regeneration capacity of limbal stem cells after exposure to MMC 0.02% with different durations in possibly in larger animal models; For an in depth investigation on these effects we are interested to see if there is any microscopic evidence of limbal stem cell loss correlating with clinical findings. Conclusion MMC may affect corneal limbal stem cells with 0.02% or lower concentrations applied topically in mouse model. We detected early signs of epithelial toxicity in the limbal region as well as signs of limbal stem cell deficiency in both concentrations if applied for 90 sec. Similar effects are suspected after refractive surgery using MMC, especially in high risk patients for development of fibrosis requiring longer durations of MMC application. References 1. 1. 2. 1. 2. Teus MA, de Benito-Llopis L, Alió JL. Mitomycin C in corneal refractive surgery. Surv Ophthalmol. 2009 Jul-Aug;54(4):487-502. Sauder G, Jonas JB. Limbal stem cell deficiency after subconjunctival mitomycin C injection for trabeculectomy. Am J Ophthalmol. 2006 Jun;141(6):1129-30. Dudney BW, Malecha MA. Limbal stem cell deficiency following topical mitomycin C treatment of conjunctival-corneal intraepithelial neoplasia. Am J Ophthalmol. 2004 May;137(5):950-1. Lichtinger A, Pe'er J, Frucht-Pery J, Solomon A. Limbal stem cell deficiency after topical mitomycin C therapy for primary acquired melanosis with atypia. Ophthalmology. 2010 Mar;117(3):431-7. Amirjamshidi H, Milani BY, Sagha HM, Movahedan A, Shafiq MA, Lavker RM, Yue BY, Djalilian AR. Limbal fibroblast conditioned media: a non-invasive treatment for limbal stem cell deficiency. Mol Vis. 2011 Mar 8;17:658-66.