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Research on mouse model of grade II corneal alkali burn
Jun-Qiang Bai,
Hai-Feng Qin, Shi-Hong Zhao
Department of Ophthalmology, Changhai Hospital, the
Second Military Medical University, Shanghai 200433, China
Co-first authors: Jun-Qiang
Bai and Hai-Feng Qin
Correspondence
to: Shi-Hong Zhao. Department of
Ophthalmology, Changhai Hospital, the Second Military Medical University, 168
Changhai Road, Shanghai 200433, China. zhaosh2001@sina.com
Received:
2015-04-06
Accepted: 2015-06-16
Abstract
AIM: To choose
appropriate concentration of sodium hydroxide (NaOH) solution to establish a stable and consistent corneal alkali burn
mouse model in grade II.
METHODS: The mice (n=60) were randomly divided into four
groups and 15 mice each group. Corneal alkali burns were induced by placing
circle filter paper soaked with NaOH solutions on
the right central cornea for 30s. The concentrations of NaOH solutions of groups
A, B, C, and D were 0.1 mol/L, 0.15 mol/L , 0.2 mol/L, and 1.0 mol/L
respectively. Then these corneas were irrigated with 20 mL physiological saline (0.9% NaCl). On day 7 postburn, slit lamp microscope was used to
observe corneal opacity, corneal epithelial sodium fluorescein staining
positive rate, incidence of corneal ulcer and corneal neovascularization,
meanwhile pictures of the anterior eyes were taken. Cirrus spectral domain
optical coherence tomography was used to scan cornea to observe corneal
epithelial defect and corneal ulcer.
RESULTS: Corneal
opacity scores (
) were not significantly different between the group A
and group B (P=0.097). Incidence of
corneal ulcer in group B was significantly higher than that in group A (P=0.035). Incidence of corneal
ulcer and perforation rate in group B was lower than that in group C. Group C and D had corneal neovascularization, and
incidence of corneal neovascularization in group D was significantly higher
than that in group C (P=0.000).
CONCLUSION: Using 0.15
mol/L NaOH can establish grade II mouse model of
corneal alkali burns.
KEYWORDS: cornea;
alkali burn; mouse model; corneal neovascularization
DOI:10.18240/ijo.2016.04.02
Citation: Bai JQ,
Qin HF, Zhao SH. Research on mouse
model of grade II corneal alkali burn. Int
J Ophthalmol 2016;9(4):487-490
The alkali corneal burn
model, an animal model of ocular surface diseases, is
commonly used to study pathological and physiological changes induced by
injuries. The basic method is putting circle filter paper (2-mm diameter)
soaked NaOH solution on the central cornea, and different concentrations of
NaOH solution cause different damages to the cornea. Reports
show, most of grade I
corneal burns can be healed well without treatments, most
of grade
II corneal burns
irrigated timely can be healed well[1-6].
When being used to treat grade II corneal burns,
diphoterine solution has significantly faster healing effect than physiological saline.
However the two irrigating solutions have no significant difference in healing
grade III
corneal burn[7-9]. Besides, the grade I corneal burn can be complete restituted,
so we should choose the animal model of corneal burn in grade II to study buffer
solutions’ therapeutic effect on corneal alkali burns. In order to establish a
mouse model of corneal alkali burn for evaluating buffer capacity of irrigating
solution, we compared corneal opacity, corneal ulcer and corneal
neovascularization of mouse’s cornea injured by NaOH solution in four different
concentrations.
MATERIALS AND METHODS
Animals All animal-based procedures were performed in accordance
with the Association for Research in Vision and Ophthalmology Statement for the
use of Animals in Ophthalmic and Vision Research, and the National Institutes
of Health Guidance for the Care and Use of Laboratory Animals. This study was
approved by the Animal Care Committee of Second
Military Medical University.
Methods Alkali
burns were inflicted in right eye of C57 mice of both sexes (n=60, male: female=1:1), 6 to 8 weeks
old, using the following protocol: mice anesthetized with 5% chloralhydrate
(0.1 mL/10 mg) intraperitoneally (IP) were randomly divided into four groups,
and were placed under the surgical dissecting microscope in a laterally
recumbent position. A single topical pontocaine 1% (ophthalmic solution) eye
drop was applied to the right cornea, followed by the filter paper (2-mm
diameter) soaked with 1.5 mL
NaOH solution for 30s. The concentrations of NaOH solutions for group A, group
B, group C, group D were 0.1
mol/L, 0.15 mol/L, 0.2 mol/L and 1.0 mol/L respectively. After the filter paper was removed from the cornea, the
eye was thoroughly irrigated with 20 mL sterilized physiological saline.
Mice were housed with plastic cages, normal diet, with 12/12h day and night.
The slit lamp microscope (SLM-3, Chongqing Kang Hua technology co., Chongqing,
China) was used to observe cornea, conjunctiva, the anterior chamber
every day and the results of observation were recorded. On
day 7, mice were anesthetized and we used camera attached to the slit lamp
microscope to watch corneas and take pictures,
meanwhile, Cirrus spectral domain optical coherence tomography
(Germany Zeiss Co., Germany)
was used to scan the anterior of the eye. Mice were sacrificed with overdose 5%
chloral hydrate at the end of experiment.
Mesurement After corneal
alkali burn, two researchers (Bai JQ and Qin HF) used the slit-lamp microscope
(magnify: ×10, angle: 45°, brightness: 5, Kanghua SLE imaging
soft V1.1) to observe corneas, conjunctivas, anterior chambers and
symblepharon, and recorded the results every day. On day
7, the researchers recorded cornea opacity scores, incidence of corneal ulcer
and corneal neovascularization, used the slit lamp microscope to take pictures,
and used Cirrus spectral domain optical coherence tomography
(Anterior Segment 5 Line Raster, spacing:
0.25 mm, lenth: 3 mm ) to scan the anterior
of eyes. As previously described[10], the degree
of cornea opacity was scored clinically on a numerical scale of 0-4: 0, clear
cornea; 1, mild stromal opacity; 2, moderate stromal opacity; 3, severe corneal
opacity with visible iris; 4, opaque cornea with iris not visible.
Exclusion
Criteria Once corneal
infection and perforation occurred, the model was considered as a failure and
excluded from statistics,
corresponding cases were added subsequently.
Statistical Analysis Statistical
analysis was performed using the statistical package SPSS v16.0 (SPSS Inc.,
Chicago, USA). Wilcoxon
signed-rank test was used to compare corneal opacity score (
). Wilcoxon signed-rank test was also used to test
corneal epithelial fluorescein staining positive rate, incidence of corneal
ulcer and neovascularization. The P
value <0.05 was considered statistically significant.
RESULTS
Morphological
Observation Immediately after central cornea injured
by alkali: groups A, B, C, D had mixed conjunctival congestion, and 2-mm
diameter circular epithelial defects with clear border can be found on the
central cornea of four groups. What’s more, corneal edema could be found in all
groups. On day 1 postburn: all the four groups still had mixed conjunctival
congestion and corneal epithelial defect of group A was partly healed , and
defect of the other three groups still clearly visible. Meanwhile, all of the
four groups still had corneal edema and group D was most edematous. On day 3,
groups C, D had neovascularization in the limbic cornea. The
neovascularization of groups C, D grew from limbic to the central cornea from
day 4 to day 7 postburn.
Corneal Opacity Score On day 7
postburn, cornea opacity scores were recorded and shown in Figure 1, Table 1.
The four groups’ corneal opacity scores (M±QR) had same QR (QR=1). Corneal
opacity score () among four groups were significantly different (P=0.000): corneal opacity scores () was not significantly different between the group A and
group B (P=0.097); corneal opacity
scores () of group C and group D significantly higher than that
of group A (P=0.008, and P=0.000, respectively ); corneal opacity
scores () was not significantly different between group B and
group C (P=0.124); corneal opacity
scores () of group D significantly higher than that of group B (P=0.000); corneal opacity scores () of group D significantly higher than that in group C (P=0.000).
Table 1 Corneal opacity scores on day 7
n=15
|
Groups |
Corneal opacity scores |
||||||
|
0 |
1 |
2 |
3 |
4 |
M±QR |
|
|
|
A |
4 |
8 |
3 |
0 |
0 |
1±1 |
0.87±0.726 |
|
B |
0 |
10 |
5 |
0 |
0 |
1±1 |
1.33±0.477 |
|
C |
0 |
6 |
8 |
1 |
0 |
2±1 |
1.67±0.477 |
|
D |
0 |
0 |
3 |
9 |
3 |
3±1 |
2.87±0.726 |
1Wilcoxon signed-rank test.
Corneal Epithelial Sodium Fluorescein Staining Positive Rate, Incidence of
Corneal Ulcer and Corneal Neovascularization Results
of corneal epithelial sodium fluorescein staining positive rate, incidence of
corneal ulcer and corneal neovascularization on day 7 were showed in Figures 1,
2, 3, and 4. Corneal epithelial sodium fluorescein staining positive rates were
not significantly different between four groups (P>0.05). Incidences of corneal ulcer in four groups were
significantly different (P=0.000):
incidence of corneal ulcer in group B was significantly higher than that in
group A (P=0.035, Fisher); incidences
of cornea ulcer in group C and group D were significantly higher than that in
group A (P=0.001, and P=0.000, respectively),
incidence of corneal ulcer in group C was significantly higher than in group B
(P=0.020), incidence of corneal ulcer in group D was
significantly higher than that in group B (P=0.002,
Fisher); incidence of corneal ulcer in group D was significantly higher than
that in group C (P=0.0483). Incidence
of corneal neovascularization in group D was significantly higher than that in
group C (P=0.000).
Figure 1 The anterior imaging using the slit lamp
microscope on day 7 Presence of corneal opacity and corneal
neovascularization: corneal opacity scores of A, B, C and
D are 0, 1, 2 and 4 respectively, and D has corneal neovascularization.
Figure 2 Corneal fluorescein staining image
using the slit lamp microscope on day 7
Presence of corneal
epithelial defect and corneal ulcer: pictures of A and B have corneal epithelial defect, pictures of C and D
have corneal ulcer.
Figure 3 Cirrus spectral domain optical coherence tomography (spacing:
0.25 mm, lenth: 3 mm ) scan cornea of mouse eye on day 7 Presence of corneal epithelial defect and corneal ulcer: pictures of A and B have corneal epithelial defect,
pictures of C and D have corneal ulcer. Central corneal thickness of A, B , C
and D are 116, 108, 96 and 80 mm
respectively; and corneal
thickness in ulcer of C and D are 60 and 68 mm.
Figure 4 Corneal
epithelial sodium fluorescein staining positive rate, incidence of corneal ulcer and corneal neovascularization
on day 7.
Mouse is an ideal
animal model, breeding economically and fast, with genome similar to human
being’s.
Mouse corneal alkali burns can result in corneal epithelial defect, corneal
ulcer, perforation of cornea, corneal opacity and corneal neovascularization.
Mouse corneal alkali burns model is an animal model used to study pathology of
corneal chemical, thermal burns, corneal neovascularization and many other
corneal diseases. According to different experiment purposes , we choose
different NaOH solutions’ concentrations, and the most commonly chosen
concentrations are 0.01, 0.1, 0.15, 1.0 mol/L [11-20]. The place putting filter paper is the central of
cornea; and corneal alkali burning times are 10, 20, 30, 40, 45 and 50s[11-20]. Filter paper sizes
reported are different, but the most often used is 2-mm diameter circle filter
paper, so filter paper used in this experiment was 2-mm filter diameter. In
some experiment, the volume of alkali used was 2 mL [13],
however, in our experiment, if 2 mL NaOH solution was dropped to 2-mm filter paper, the
excess alkali would overflow, while 1.5 mL would not. So we used 1.5 mL in this experiment finally.
According
to the system's summary: tap water, normal saline, lactated Ringer’s, normal
saline with sodium bicarbonate added, phosphate buffer solution, diphoterine
buffer can improve the prognosis of corneal burn in grades I, II (Reim 1987,
1990)[5-6]. Corneal
neovascularization are caused by alkali burn in grade III. Cornea opacity score
and ulcer incidence of group C were significantly higher than that of B group (P<0.05) on day 7, but group C has
neovascularization, which is caused by alkali burn in grade III. So we should
choose 0.15 mol/L NaOH solution to study the buffer capacity of solution and
choose 1 mol/L NaOH solution to study corneal neovascularization.
In
addition, we still need to pay attention to the following details in the
process: 1) filter paper should be placed on the central cornea, otherwise it
may cause alkali burn in non-experimental place; 2) the volume of NaOH should
be appropriate, otherwise excessive alkali may also cause alkali burn in
non-experimental place; 3) anesthetic eye drops should be used to stop
blinking; 4) the conjunctival sac should be washed thoroughly, otherwise the
occurrence of symblepharon can cause corneal injures which make it difficult to
recognize the reason for the corneal defect and corneal ulcer.
ACKNOWLEDGEMENTS
Foundation: Supported by National Science and Technology
Major Project of China (No. 2011ZXJ09104-10C).
Conflicts of Interest: Bai JQ, None; Qin HF, None; Zhao SH, None.
REFERENCES [Top]
1 Beare JD. Eye injuries from assault
with chemicals. Br J Ophthalmol
1990;74(9):514-518. [CrossRef]
2 Wagoner MD. Chemical injuries of the
eye: current concepts in pathophysiology and therapy. Surv Ophthalmol 1997;41(4):275-313. [CrossRef]
3 Kuckelkorn R, Schrage N, Keller G,
Redbrake C. Emergency treatment of chemical and thermal eye burns. Acta Ophthalmol Scand 2002;80(1):4-10. [CrossRef]
4 Fish R, Davidson RS. Management of
ocular thermal and chemical injuries, including amniotic membrane therapy. Curr Opin Ophthalmol 2010;21(4):317-321.
[CrossRef]
5 Schrage NF, Struck HG, Gerard M.
Recommendations for acute treatment for chemical and thermal burns of eyes and
lids. Ophthalmologe 2011;108(10):916-920. [CrossRef] [PubMed]
6 Chau JP, Lee DT, Lo SH. A systematic
review of methods of eye irrigation for adults and children with ocular
chemical burns. Worldviews Evid Based
Nurs 2012;9(3):129-138. [CrossRef] [PubMed]
7 Cho WK, Kang S, Choi H, Rho CR.
Topically administered gold nanoparticles inhibit experimental corneal
neovascularization in mice. Cornea 2015;34(4):456-459. [CrossRef] [PubMed]
8 Iakimenko SA, Buznyk OI,
Rymgayllo-Jankowska B. Amniotic membrane transplantation in treatment of
persistent corneal ulceration after severe chemical and thermal eye injuries. Eur J Ophthalmol 2013;23(4):496-503. [CrossRef] [PubMed]
9 Merle H, Donnio A, Ayeboua L, Ayeboua
L, Michel F, Thomas F, Ketterle J, Leonard C, Josset P, Gerard M. Alkali ocular
burns in Martinique (French West Indies) Evaluation of the use of an amphoteric
solution as the rinsing product. Burns 2005;31(2):205-211. [CrossRef] [PubMed]
10 Yoon KC, Heo H, Kang IS, Lee MC, Kim
KK, Park SH, Cho KO. Effect of topical cyclosporine A on herpetic stromal
keratitis in a mouse model. Cornea 2008;27(4):454-460.
[CrossRef] [PubMed]
11 Yan J, Zeng Y, Jiang J, Zhou J, Yin
Z, Wang Z, Zhu P. The expression patterns of vascular endothelial growth factor
and thrombospondin 2 after corneal alkali burn. Colloids Surf B Biointerfaces 2007;60(1):105-109. [CrossRef] [PubMed]
12 Xiao O, Xie ZL, Lin BW, Yin XF, Pi
RB, Zhou SY. Minocycline inhibits alkali burn-induced corneal
neovascularization in mice. PLoS One
2012;7(7): e41858. [CrossRef]
[PubMed] [PMC free article]
13 Kubota M, Shimmura S, Kubota S,
Miyashita H, Kato N, Noda K, Ozawa Y, Usui T, Ishida S, Umezawa K, Kurihara T,
Tsubota K. Hydrogen and
N-acetyl-L-cysteine rescue oxidative stress-induced angiogenesis in a mouse
corneal alkali-burn model. Invest
Ophthalmol Vis Sci 2011;52(1):427-433. [CrossRef] [PubMed]
14 Zhou Q, Yang L, Qu M , Wang Y, Chen
P, Wang Y, Shi W. Role of senescent fibroblasts on alkali-induced corneal
neovascularization. J Cell Physiol
2012;227(3):1148-1156. [CrossRef]
[PubMed]
15 Cade F, Paschalis EI, Regatieri CV,
Vavvas DG, Dana R, Dohlman CH. Alkali burn to the eye: protection using TNF-α
inhibition. Cornea 2014;33(4):382-389.
[CrossRef] [PubMed]
16 Giacomini C, Ferrari G, Bignami F,
Rama P. Alkali burn versus suture-induced corneal neovascularization in C57BL/6
mice: an overview of two common animal models of corneal neovascularization. Exp Eye Res 2014;121:1-4. [CrossRef] [PubMed]
17 Sakimoto T, Sugaya S, Ishimori A,
Sawa M. Anti-inflammatory effect of IL-6 receptor blockade in corneal alkali
burn. Exp Eye Res 2012;97(1):98-104. [CrossRef] [PubMed]
18 Zhou WJ, Liu GQ, Li LB, Zhang XG, Lu
PR. Inhibitory effect of CCR3 signal on alkali-induced corneal
neovascularization. Int J Ophthalmol 2012;5(3):251-257. [PMC free article]
[PubMed]
19 Wang Y, Yin H, Chen P, Xie L, Wang Y.
Inhibitory effect of canstatin in alkali burn-induced corneal
neovascularization. Ophthalmic Res
2011;46(2):66-72. [CrossRef]
[PubMed]
20 Chen P, Yin H, Wang Y, Mi J, He W,
Xie L, Wang Y. Multi-gene targeted antiangiogenic therapies for experimental
corneal neovascularization. Mol Vis 2010;16:310-319.<bb>
[PMC free article]
[PubMed]
[Top]