The Effect of Ascorbic Acid (Vitamin C)

on Transepithelial Corneal Cross-Linking in

Rabbits
Mustafa Koc, Basak Bostanci, Ozlem Ozbas Demirel,Feyza Genc, Kemal Tekin,
Yaran Koban,Aylin Sepici Dincel, Murat Sen, and Pelin Yilmazbas

Presented by :
dr. Ezra Margareth
dr. Noor Aminah
dr. Teguh Setiawan
ABSTRACT
• Purpose: To evaluate the effects of ascorbic acid (vitamin C), the main
antioxidant agent in the cornea on transepithelial corneal cross-linking
(CXL) where the main mechanism is oxidation.
• Methods: Twenty eyes of 20 rabbits were divided into 3 groups: Group 1 (7
eyes) had transepithelial corneal CXL after being fed with normal diet;
Group 2 (7 eyes) had corneal CXL after once-daily subcutaneous injections
of 200 mg of ascorbic acid in addition to normal diet; and the control group
(6 eyes) was fed with normal diet but did not have corneal CXL performed.
Ascorbic acid levels were measured in aqueous humor and plasma, and
biomechanical measurements were applied to the cornea.
• Results: There was a significant difference in ascorbic acid levels of plasma
and aqueous humor between group 1 and 2. The Young’s modulus values of
group 1 and 2 were similar and were significantly higher than the control
group.
• Conclusions: Systemic vitamin C does not appear to decrease
effectiveness of transepithelial corneal CXL. Therefore, there is no reason to
stop or reduce vitamin C supplementation before corneal CXL therapy.
INTRODUCTION
• Corneal cross-linking (CXL) is the only treatment that
can halt the progression of keratoconus.
• Riboflavin interacts with ultraviolet-A (UVA) and
produces free radicals and reactive oxygen species into
the corneal stroma.
• This photo-oxidative reaction induces formation of
additional covalent bonds between the collagen fibers
and increases the biomechanical stability of the cornea.
• There is no 1 corneal CXL protocol and various
individual protocols have been published.
• In the most commonly applied conventional corneal CXL
protocol, corneal epithelium is removed (epi-off CXL)
INTRODUCTION
• The removal of epithelium decreases patient
comfort, delays visual rehabilitation, and increases
the likelihood of complications such as ulceration,
haze, and infection.
• Various protocols have been developed to enhance
riboflavin permeability without removing the
epithelium, such as the use of iontophoresis,
benzalkonium chloride, ethylenediamine tetraacetic
acid (EDTA), and tromethamine (transepithelial =
epi-on CXL).
• Most studies found that the effectiveness of
transepithelial corneal CXL is lower than that of epi-
off corneal CXL.
INTRODUCTION
• The positioning of the eye  constantly exposed to high
amounts of UV radiation.
• The most important antioxidants in the eye against reactive
oxygen species formed due to UV exposure are glutathione
and ascorbic acid.
• Glutathione : found in the lens
• Ascorbic acid (vitamin C) : found in the corneal epithelium.
Ascorbic acid is not synthesized and stored in the human
body; therefore, dietary intake is essential.
• Corneal CXL is dependent on oxidation, and ascorbic acid is
one of the most important antioxidant molecules in the
corneal epithelium and commonly used dietary supplements
• This study aim to determine whether ascorbic acid affects
transepithelial corneal CXL in rabbit models.
METHODS
• Animal preparation
• Sample : Twenty eyes of 20 New Zealand rabbits, aged 4–6 months and
weighing 3–5 kg
• All experiments were performed in accordance with the Association for
Research in Vision and Ophthalmology Resolution on the Use of Animals in
Research and approved by Ankara University Ethical Committee on Animal
Research.
• The rabbits were randomly assigned to 3 groups

• 7 rabbits was given a • 7 rabbits was given • the control group (6

normal diet for 15 the same diet along rabbits) was given the
days with once-daily normal diet for 15
subcutaneous days
injections of 200 mg
of ascorbate
(Redoxon!) for 15 days
METHODS
• Animal preparation
• On the 16th day  the rabbits were premedicated with intramuscular injection of
xylazine (5 mg/kg) and anesthetized by intramuscular injection of ketamine
hydrochloride (35 mg/kg).

• Using a 26-gauge needle, 50 mL of aqueous humor drawn from the anterior

chamber and 2mL of blood was withdrawn from the superior auricularis vein of each
of the rabbits in group 1 and 2.

• Aqueous humor and plasma samples from the control group were not evaluated for
ascorbic acid levels since the control group was given a similar diet to group 1.
• All of the rabbits in group 1 and 2 were rested for 2 h to allow the anterior chambers
to re-form, then we performed transepithelial corneal CXL.
 METHODS
• Corneal cross-linking
The thickness of the central cornea was measured with an ultrasonic pachymeter
(UP-1000; Nidek Co., Ltd.). Isoosmolar 0.1% riboflavin solution (Ricrolin TE!;
Sooft, Italy), containing EDTA and tromethamine, was applied to the cornea every
2 min for 30 min in group 1 and 2. Afterward, UVA irradiation (3 mW/cm2,
Apollon Crosslinking System!; Meran Tıp, Turkey) was performed for 30 min.

Researcher continued to drop riboflavin solution during irradiation at the same

frequency. The control group received the riboflavin drops for 60 min but was not
irradiated. Following CXL, the rabbits were sacrificed by intra-cardiac injection of
150 mg/kg sodium pentobarbital.
 METHODS
Specimen preparation and biomechanical
measurements

The scleral ring was cut  4 x 10mm vertical corneal strip was
removed from the superior-inferior position.

Corneal biomechanical properties measured using a microcomputer-controlled

biomaterial tester (Zwick Z010, Ulm, Germany) and the strip extensiometry
method. The corneal strip was clamped vertically at a distance of 5mm between
the jaws of a biomaterial testing device.

A preload power of 20mN was applied to the strip before the test. The strain
was increased linearly at a rate of 1 mm/min and was measured up to tissue
rupture.

The parameters of Young’s modulus, ultimate stress, and ultimate strain were
determined and used for data analysis. Young’s modulus values (relation
between tangential force and cross-sectional area) were calculated for 10%
strains from the stress-strain curves by TestXpert software.
METHODS
• Ascorbic acid measurement
• Blood samples were immediately cooled on ice and
plasma was removed by centrifugation at 2,000 g for 10
min at 4"C.
• Meta-phosphoric acid was added to the plasma and
aqueous humor samples that were then centrifuged at
10,000 g for 5 min at 4"C.
• The supernatants were removed and stored in the dark
at -70"C until analysis. The samples were thawed,
filtered, and injected directly on to a High Performance
Liquid Chromatography system (HPLC, NH2 column),
with measurements taken at 254 nm.
METHODS
• Statistical analysis
• The data were analyzed using SPSS software,
version 20 (IBM, Armonk, New York). Statistical
significance was determined by ANOVA-Tukey
and Mann–Whitney tests, with P values <0.05
considered to be significant.
RESULT

• The mean central corneal thicknesses were similar between

the groups: 398 – 31 mm for group 1, 414 – 28 mm for group
2, and 406 – 30 mm for control group (P > 0.05 for all
groups).
RESULT
• tabel
• thankyou