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

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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
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