31

Original Investigation Abstract

Objective: To evaluate our modifed corneal alkali rat model and compare with the Ma et al. corneal alkali model.
Methods: Corneal alkali injury by the method of Ma et al. was applied to 10 rats in Group 1 and corneal alkali injury with
our modifed method was applied to 10 rats in Group 2. Hypopyon, hyphema, corneal fstula, phthisis bulbi occurrence
and expected epithelial damage success rate and ultrastructural changes were evaluated in both groups.
Results: Phthisis bulbi incidence was higher in Group 1 and expected epithelial damage success rate was higher in
Group 2. Histological analysis revealed that stability of basal layers of epithelium of cornea was much more disturbed
in Group 1.
Conclusion: Our modifed corneal alkali injury animal model is an efective animal model with an increased epithelial
success rate and decreased incidence of phthisis bulbi.
Key words: Corneal alkali burn, Rat model
Niche, 2012; 1: 31-3 doi : 10.5152/niche.2012.07
A Modifed Rat Model for Corneal
Alkali Burn
Emrullah Beyazyldz
1
, Ferda Alpaslan Pnarl
2
, zlem Beyazyldz
2
, mit Beden
3
,
Tuncay Deliba
3
1
Gazi Mustafa Kemal State Hospital,
Department of Ophthalmology, Ankara,
Turkey
2
Dskap Yldrm Beyazt Research and
Training Hospital, Clinic of Cell Research and
Genetic Diagnosis Center, Ankara, Turkey
3
Ulucanlar Eye Research and Training
Hospital, Department of Ophthalmology,
Ankara, Turkey
4
Memorial Ataehir Hospital, Department of
Ophthalmology, stanbul, Turkey
5
Dskap Yldrm Beyazt Research
and Training Hospital, Department of
Endocrinology and Metabolism, Ankara,
Turkey
Submitted : 17.10.2012
Accepted : 26.01.2012
Correspondence: Dr. Emrullah Beyazyldz
Clinic of Ophthalmology, Gazi Mustafa Kemal
Government Hospital, Ankara, Turkey
Phone: +90 312 271 83 58
E-mail: dremrullah@hotmail.com
Copyright 2012 by Cellular Therapy and
Regenerative Medicine Society
Available online at www.nichejournal.org
Introduction
Chemical exposure to the external eye can result
in trauma, ranging from mild irritation to severe
damage to both the ocular surface and anterior
segment, leading to permanent vision loss. Severe
damage occurs when alkali chemicals penetrate
the cornea within seconds, leaving a white non-
transparent corneal opacity (1).
There is a wide range of therapeutic approaches
used to treat alkali eye injuries. However, more ef-
fective strategies are still required to treat severe
cases, and new treatment modalities such as stem
cell-based therapeutics are being studied in cor-
neal alkali burn animal models.
The Ormerod et al. (2) animal model is widely ac-
cepted and commonly used for corneal alkali burn
research. However, although this model is consid-
ered the standard for corneal alkali burn, severe
corneal ulcers with hypopyon, hyphema, corneal
fstula and phthisis bulbi have been reported in
several studies using this model (3). Ma et al. (3)
modifed the Ormerod et al. (2) method, but still
observed high levels of excessive corneal burn
rates. Therefore, for these studies, we sought to
modify the corneal alkali injury model in an efort
to decrease excessive corneal burn rates and other
complications associated with other models , spe-
cifcally comparing the results from our modifed
corneal alkali rat model with the model employed
by Ma et al. (3).
Methods
Animals
This study was approved by the Animal Research
Ethics Committee of the Dkap Yldrm Beyazt
Training and Research Hospital, and the study
was undertaken at the Experimental Animal Re-
search Center of Dkap Yldrm Beyazt Training
and Research Hospital. Animal experiments were
performed in a manner consistent with the ARVO
Statement for the Use of Animals in Ophthalmic
and Vision Research. Twenty Spraque-Dawley rats
(200-250 g) were used in this study. Rats were anes-
thetized by ether inhalation. The experiment was
carried out on the right eye of each animal. At the
end of the experiment, all rats were sacrifced with
an overdose of 10% chloral hydrate and corneas of
the rats were harvested for histological analysis.
Grouping and Corneal Injury Methods
For these studies, rats were randomly divided into
two groups: 10 rats were subjected to the Ma et al.
(3) model of corneal alkali injury (Group 1) and 10
rats received our modifed model of corneal alkali
32
Beyazyldz et al. A Modifed Rat Model for Corneal Alkali Burn Niche 2012; 1: 31-3
injury (Group 2). Briefy, for Group 1, a flter paper ring (ID 4 mm
and OD 8 mm) was saturated with 1 N NaOH and applied to the
center of the cornea for 30 s. The cornea was then rinsed with
saline (60 mL) for 20 s. In animals from Group 2, a flter paper ring
(ID 4 mm and OD 8 mm) saturated with 1 N NaOH was applied to
the center of the cornea for 20 s. The cornea was then rinsed with
saline (60 mL) for 10 s. Following the corneal alkali burn, animals
from both groups were treated with gentamicin and dexametha-
sone eye drops twice a day for one week (Genta oft 0,35% -Bilim
drug, Dexa-sine 5 mL-Liba medical).
Clinical Outcome and Analysis
Following corneal injury, the eye of each animal was monitored
daily for hypopyon, hyphema, corneal fstula and phthisis bulbi
development. Corneas were also examined to see whether there
was any corneal epithelial damage throughout the entire thick-
ness of the tissue. After excision, corneas were examined under a
direct light microscope to assess corneal clarity and neovascular-
ization. Corneal transparency was evaluated as: completely trans-
parent, iris not clear, or pupil could not be seen. Corneal neovas-
cularization was evaluated as: no neovascularization, detected
within 2 mm from the limbus, or detected over 2 mm from the
limbus. Corneal epithelial defects were assessed in tissue stained
with fuorescein using a microscope. Corneal epithelial defects
were evaluated as: staining more than 50% of whole cornea or
less than 50% of whole cornea (Figure 1).
Histological Analysis
Excised corneas were collected, fxed in 10% paraformaldehyde,
and embedded in parafn. Preparations with 5 m sections were
stained with hematoxylin and eosin (H&E). Preparations were an-
alyzed using a Leica HMLB45 light microscope (Leica, Germany).
Statistical Analysis
The SPSS 13.0 program was used for statistical analysis. The Pear-
sons Chi-square test, Fischer exact test and continuity-adjusted
Chi-square tests were used. A probability level of p<0.05 was con-
sidered signifcant.
Results
Following the surgical procedure, infection, hyphema, and cor-
neal fstula incidence were assessed. No signifcant diferences
were observed between the two groups (p>0.05). Infection and
corneal fstula were observed in 1 rat from Group 1 but hyphema
did not occur in animals from either group. Phthisis bulbi inci-
dence and expected epithelial damage success rate were com-
pared between the two models of injury. Phthisis bulbi incidence
was signifcantly greater in Group 1 (50%) when compared with
Group 2 (10%) (p<0.05). In contrast, expected epithelial success
rate was signifcantly greater in Group 2 (70%) compared with
Group 1 (30%) (p<0.05). A summary of all measurements can be
found in Table 1.
Corneal epithelium of the rats were histologically analyzed. The
corneal epithelial basal layer was separated from the stroma and
stability of the cornea was disrupted in Group 1 and basal layers of
the corneal epithelium remain attached to stroma of cornea and
stability in corneal epithelial basal layers was preserved in Group
1. Histological analysis showed greater disruption to the stabil-
ity of the basal layers of cornea epithelium in rats from Group 1
(Figure 2) when compared with those in Group 2 (Figure 3).
Discussion
Alkali injuries constitute one of the most urgent ocular emer-
gencies and are notoriously destructive due to their rapid pen-
etration of ocular tissues. Alkali injuries can change the corneal
transparency, resulting in a dramatic decrease in visual acuity or
Figure 1. Evaluation of rat cornea stained with fuorescein using a co-
balt blue-light under the microscope. Cornea epithelial damage oc-
curred in the absence of phthisis bulbi
Figure 2. Histological analysis (H&E stain, x200) of rat cornea alkali
burn generated with Ma. et. al. (3) model. Disruption in corneal epi-
thelial basal layers is clearly observed. Corneal epithelial basal layer is
separated from the stroma and stability of cornea is disrupted
Figure 3. Histological analysis (H&E stain, x200) of rat cornea alkali burn
generated with our modifed model. Basal layers of cornea epithelium
remain attached to stroma of cornea and stability in corneal epithelial
basal layers is preserved
33
Beyazyldz et al. A Modifed Rat Model for Corneal Alkali Burn Niche 2012; 1: 31-3
complete loss of sight. Corneal ulceration, perforation and neo-
vascularizations are the most common acute complications of
corneal chemical burns. Thus acute management of corneal al-
kali burns is crucial for successful clinical treatment of the injury
(4). Although there are a number of therapeutic modalities for
the treatment of alkali burn, to date, no ideal method is available,
especially for severe burns. Recent studies have investigated the
efects of mesenchymal stem cells in animal models of corneal
alkali burn, but further tests are needed to elucidate the poten-
tial therapeutic efcacy of mesenchymal stem cells. For the cur-
rent study, we aimed to fnd a more efective corneal alkali burn
rat model to decrease the number of animal loss. This should be
particularly useful for future studies testing new potential thera-
peutic strategies.
A standardized corneal alkali injury animal model was frst devel-
oped by Ormerod et al. (2). Before this, corneal burn was only pro-
duced using an alkali-flled well applied to the corneal surface (5).
After a variable period of time, the well was drained and removed
and the eye irrigated. However, it was difcult to produce uniform
lesions with this method. Ormerod et al. (2) achieved a central dis-
coid corneal burn in rabbits which permitted clinical evaluation
of the corneal neovascular response. They used 7 mm flter paper
discs which were soaked for 10-20 s in 1 N NaOH and applied to
the cornea for 2 min. Ye et al. (6) studied a corneal alkali injury
model using 3 mm diameter flter paper soaked with 1 N NaOH
and applied to the center of the cornea for 40 s; followed imme-
diately by 1 min of corneal rinsing. They did not report any inci-
dence of corneal ulcer, hypopyon or hyphema. However, when
Ma et. al. (3) used identical methods, they reported corneal ulcer,
hypopyon, hypema, corneal fstula and severely damaged corne-
al stroma . Even with a modifed method using a flter paper ring
(ID 4 mm and OD 8 mm), applied to the cornea for 30 s, and then
immediately rinsed with saline 20 s, Ma et al. (3) still observed
corneal ulcers and other surgical complications. For the current
study, we used the method of Ma et al. (3) for Group 1, and phthi-
sis bulbi occurred in 50% of rats, along with a lower epithelial
success rate. For Group 2, we utilized a modifed method which
decreased both the NaOH application time (20 s) and the rins-
ing time (10 s). With these modifcations, phthisis bulbi incidence
was decreased to 10% and epithelial success rate was increased
to 70%.
Conclusion
These results suggest that our modifed corneal alkali injury is
an efective animal model for inducing corneal injury with an in-
creased epithelial success rate and decreased incidence of phthi-
sis bulbi, and could be useful for future therapeutic studies.
Conflict of interest: No conflict of interest was declared by the
authors.
References
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[CrossRef]
2. Ormerod LD, Abelson MB, Kenyon KR. Standard models of corneal
injury using alkali-immersed filter discs. Invest Ophthalmol Vis Sci
1989; 30: 2148-53.
3. Ma Y, Xu Y, Xiao Z, Yang W, Zhang C, Song E, et al. Reconstruction
of chemically burned rat corneal surface by bone marrow-derived
human mesenchymal stem cells. Stem Cells 2006; 24: 315-21.
[CrossRef]
4. William Tasman Duanes Ophthalmology. 2006 edition Philadelphia:
Lippincott Williams & Wilkins, 2006.
5. Pfister RR, Burstein N. The alkali burned cornea I. Epithelial and
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Hyphema Corneal Phytisis Expected epithelial
(n-%) Fistula Bulbi damage
(n-%) (n-%) (n-%)
Group 1 0 (0) 1 (10) 5 (50) 3 (30)
Group 2 0 (0) 1 (10) 1 (10) 7 (70)
Table 1. Comparison of groups according to infection, hyphema,
corneal fstula, phytisis bulbi and expected epithelial damage rates