|
In Vitro Effects of
Ascorbic Acid on Corneal Collagen Cross-Linking
in Keratoconus
Nasrin Aghaei (1)
Shahrokh Ramin (2)
Abbas Aghaei (3)
Sayed Mehdi Tabatabaei (4)
Mohammd Aghazadeh Amiri (5)
(1) MSc student in Optometry, Department
of Optometry, School of Rehabilitation, Shahid
Beheshti University of Medical Sciences, Tehran,
Iran.
(2) Ophthalmologist, Department of Optometry,
School of Rehabilitation, Shahid Beheshti University
of Medical Sciences, Tehran, Iran.
(3) PhD student in Epidemiology, Research Development
Center of Imam Khomeini Hospital, Kermanshah
University of Medical Sciences, Kermanshah,
Iran.
(4) PhD student in Biostatistics, School of
Rehabilitation, Shahid Beheshti University of
Medical Sciences, Tehran, Iran.
(5) OD of Optometry, Department of Optometry,
School of Rehabilitation, Shahid Beheshti University
of Medical Sciences, Tehran, Iran.
Correspondence:
Shahrokh Ramin,
Department of Optometry,
School of Rehabilitation,
Shahhid Beheshti University of Medical Sciences,
Tehran,
Iran
Email: s.ramin@sbmu.ac.ir
|
Abstract
Purpose: To
assess the efficacy and safety of ascorbic
acid in the treatment of keratoconus by
increasing the number of “anchors”
that bond collagen fibers together in
human in vitro cornea using electron microscopy.
Methods:
In this semi experimental study keratoconus
cornea is divided into six equal parts
after keratoplasty. Two doses of ascorbic
acid (10-3 mg/ml and 10-4 mg/ml) in two
time ranges of 4 hours and one week after
the treatment are applied to analyze the
deviations of cross-linking. A part of
the cornea is considered as control sample
(without ascorbic acid) for different
times. Various parts are randomly assigned
to different studied doses. Each section
of cornea is evaluated using electron
microscopy. Friedman and Wilcoxon tests
are used for data analysis. The value
of significance level was set at 0.05.
Results:
The average distance between collagen
fibrils are measured after the treatment
with two different doses of ascorbic acid
for two different time ranges. These results
showed that higher doses of ascorbic acid
and longer treatment time led to lower
distance between collagen fibrils of cornea
(p <0.001). This implies the better
strength of the cornea. Apoptosis and
vacuolization were not observed in keratocytes
by electron microscopy after treatment
with ascorbic acid.
Conclusions:
Results showed that ascorbic acid
strengthens the cornea and decreases the
distance between collagen fibrils (consequently
increase cross-linking). Therefore, the
efficacy of ascorbic acid is observed
by more recovery through increasing its
doses and passing time.
Key words:
Ascorbic acid, Cross-linking, Cornea,
Keratoconus, Keratocytes
|
Cornea is the anterior and transparent segment
of the eyeball. There are five layers from anterior
to posterior of cornea: epithelium, bowman,
stroma, Descemet’s membrane and the endothelium.
Stroma, constitutes 90% of corneal thickness,
and consists of collagen fibrils blades intussusception
(anchors). Keratocytes, as important formation
cells of stroma, produce collagens and their
apoptosis and necrosis cause structural weakness
of corneal tissues (1).
Keratoconus (KC) is a bilateral, progressive,
degenerative and non-inflammatory pathology
of corneal ectasia(2). It usually starts at
puberty. Irregular astigmatism, corneal thinning
and progressive myopia and protrusion are characteristics
of keratoconus that lead to decreased visual
acuity(3). The prevalence of KC in the general
population is reported to be around 1/2000 often
affecting young patients(4). The etiopathogenesis
and pathogenesis of keratoconus is unknown but
it is likely a multifactorial disease (5). There
are changes observed in corneal collagen organization
(6), structure (7), intercellular matrix, necrosis
and apoptosis of keratinocytes (8). Furthermore,
a reduction is observed in cross-linking chemical
bonds between collagen fibers inside the stroma
for keratoconic corneas as compared to normal
ones (9). This implies structurally weakened
keratoconus corneal tissue (8).
There was not any effective way to stop progressive
keratoconus so that about 21% of keratoconus
patients need corneal transplantation (10).
Conventional methods only improve the refractive
errors and the loss of vision of corneal ectasia
such as spectacles, rigid contact lenses, scleral,
mini scleral and hybrid lenses, photorefractive
keratectomy, intrastromal corneal ring, epikeratoplasty,
phakic intraocular lens, keratophakia, keraflex
(11) and penetrating keratoplasty. It should
be noted that these methods do not remedy keratoconus
and cannot stop the progression of this disease
(2, 12).
Corneal collagen cross-linking is introduced
for the treatment of progressive keratoconus(13).
The first research of photobiology was conducted
to increase the resistance of stromal collagen
in the 1990s (14). The procedure of corneal
collagen cross-linking is a combined action
of chemical and physical factors which consisted
of UVA ray at 370 nm (15) and a photosensitizing
substance (riboflavin or vitamin B2) (8). Corneal
rigidity is increased by corneal collagen cross-linking
(16). This treatment is based on the changes
of biomechanical properties of stromal collagen
and cause pathogenesis of keratoconus (10).
Investigations show that oxidative stress is
effective in KC (5, 17). Oxidative stress leads
to disorder in the normal state of the cell
and destruction of all cellular components including
proteins and collagens resulted in a toxic effect
by free radicals (18). Vitamin C (ascorbic acid)
and vitamin E (-tocopherol) react with free
radicals. It is strongly accepted that the antioxidant
virtues of these vitamins are responsible for
their biological activity (18). Ascorbic acid
is a basis for normal collagen formation (19).
It is a main component in the synthesis of hydroxylysine
and hydroxyproline in collagen (20). Hydroxylysine
is important for formation of the intermolecular
collagen cross-linking (21). Furthermore, ascorbic
acid plays an important role in collagen biosynthesis
(22, 23).
In this study, the distance between collagen
fibrils are measured because of the study of
cross-linking was qualitative and there were
no plans or software to count cross-linking.
The consistency of cornea is increased by reducing
the distance between collagen fibrils. The purpose
of this pilot study was to assess the efficacy
and safety of ascorbic acid in the treatment
of keratoconus by increasing the number of “anchors”
that bond collagen fibers together (the corneal
cross-linking strengthen the cornea). This study
can be considered as a basis for the future
conservative treatment of keratoconus.
This semi experimental study was performed
on patients attending the cornea clinic at Negah
hospital in Tehran, Iran. The participants of
this study were patients with confirmed diagnosis
of progressive keratoconus who had an operation
of penetrating keratoplasty. Two keratoconus
corneal buttons (7-8.5mm) were obtained from
25 and 26-year-old patients treated with 10-3
and 10-4 mg/ml ascorbic acid (chemical formula:
C6H8O6, molecular weight (MW): 176.13 gr/mol
and Art No: F413727). Two corneal buttons were
sliced individually with a microtome to six
equal sections. One section was stored in optisol
and 10-3 mg/ml ascorbic acid. Another section
was stored in optisol and 10-4 mg/ml ascorbic
acid for 4 hours. Furthermore, one randomized
section was just soaked in optisol as a control
sample. Three samples of cornea were considered
to be examined after one week as witness groups:
one at optisol with 10-3 mg/ml ascorbic acid,
one at optisol with 10-4 mg/ml ascorbic acid
and the last one at optisol.
Transmission Electron Microscopy
After the treatment with ascorbic acid, corneal
buttons with their controls were prepared for
transmission electron microscopy (TEM) (Zeiss
EM 900). All sections were immersed in 2% glutaraldehyde.
Sections of corneal button were embedded in
hard resin after being fixed and dehydrated
for transmission electron microscopy. 50–70-nm
ultrathin sections were prepared and mounted
on copper grids when the semithin sections stained
with toluidine blue were observed.
Grids containing color samples were prepared
to observe by transmission electronmicroscopy.
EMC software V.16.8 was applied to study respective
areas. In order to select images: magnifications
of 7000 and 12000 were used to assess keratocytes
(vacuolization and evaluation keratocyte membrane),
the magnification of 30,000 was applied to assess
the distance between collagen fibrils. 150000
sections of each sample were prepared by microkeratome
and about 30 images of each section was taken.
Finally,
10 images were selected to analyze keratocytes,
cross-linking and the distance between collagen
fibrils.
The distance between collagen fibrils was measured
by AutoCAD 2007 software. In each case, the
distance between the fibrils was randomly measured
at 100 points to obtain an average. The average
of different 100 points were randomly obtained
for three cases which were almost same without
any considerable difference. Images were passed
to two medical geneticists to study keratocytes
according to apoptosis, necrosis, the existence
of vacuolization in them, shrinkage and freshness.
They separately commented on freshness and keratocytes
apoptosis rate in different doses of ascorbic
acid at different times of treatments. It should
be mentioned that the mean comments of these
two medical geneticists are reported.
Considering that the control and treated groups
were prepared from two corneas and comparisons
between samples relevant only to each of these
two corneas, there was no difference between
comparisons in terms of clinical and demographic
parameters (normalized). Studied variables did
not follow a normal distribution based on the
result of Kolmogorov-Smirnov test. Therefore,
non-parametric tests were used for data analysis.
Wilcoxon test was applied to compare the interrelated
variables. Furthermore, Friedman test was used
to assess deviations over time. The first type
of error was considered about 0.05 to make decisions
about significant deviations. All statistical
analyzes were performed using SPSS v.16.
Measurements
of
the
average
distance
between
collagen
fibrils
of
two
corneas
after
the
treatment
with
two
different
doses
of
ascorbic
acid
at
4
hours
after
treatment
and
one
week
after
treatment
showed
that
increasing
the
dose
and
passing
time,
reduce
the
gap
between
collagen
fibrils
(Table
1
-
next
page).
It
should
be
noted
that
the
changes
in
the
measurement
of
control
samples
showed
the
reverse
trend.
Friedman
test
showed
a
significant
effect
of
time
on
the
change
in
distance
between
collagen
fibrils
according
to
the
comparison
of
repeated
measurements
for
the
following
conditions:
without
intervention
over
three
times,
intervention
with
10
-3
mg/ml
ascorbic
acid
at
fourth
hour,
intervention
with
10
-3
mg/ml
ascorbic
acid
in
one
week,
intervention
with
10
-4
mg/ml
ascorbic
acid
at
fourth
hour
and
intervention
with
10
-4
mg/ml
ascorbic
acid
in
one
week
(Table
2
-
page
129).
All
comparisons
are
statistically
significant
in
case
of
mutual
conditions
for
changes
in
maintenance
dose
and
time
and
also
in
comparison
with
witness
group
for
both
corneas
(Table
3-
page
130).
In
the
first
cornea,
98%
and
95%
of
keratocytes
were
healthy
and
unchanged
with
10
-3
mg/ml
and
10
-4
mg/ml
ascorbic
acid
after
4
hours,
respectively.
87%
of
keratocytes
were
healthy
and
unchanged
in
the
control
sample
after
4
hours.
78%
and
79%
of
keratocytes
were
healthy
and
unchanged
with
10
-3
mg/ml
and
10
-4
mg/ml
ascorbic
acid
after
one
week,
respectively.
72%
of
keratocytes
were
healthy
and
unchanged
in
the
control
sample
after
one
week.
In
the
second
cornea,
88%
and
92%
of
keratocytes
were
healthy
and
unchanged
with
10
-3
mg/ml
and
10
-4
mg/ml
ascorbic
acid
after
4
hours,
respectively.
83%
of
keratocytes
were
healthy
and
unchanged
in
the
control
sample
after
4
hours.
75%
and
78%
of
keratocytes
were
healthy
and
unchanged
with
10
-3
mg/ml
and
10
-4
mg/ml
ascorbic
acid
after
one
week,
respectively.
78%
of
keratocytes
were
healthy
and
unchanged
in
the
control
sample
after
one
week.
Click
here
for
Table
1:
The
distance
between
collagen
fibrils
of
cornea
for
two
distinct
patients
at
different
time
and
treatments
Click
here
for
Table
2:.Comparison
of
changes
in
measurements
during
different
times
based
on
Friedman’s
nonparametric
test
between
the
two
corneas
Click
here
for
Table
3:
Comparison
of
various
average
measurements
of
distance
between
collagen
fibrils
based
on
nonparametric
Wilcoxon
test
for
two
corneas
In
the
present
study,
we
investigated
the
effect
of
ascorbic
acid
on
the
increase
of
corneal
strength.
Significant
changes
in
the
distance
between
collagen
fibrils
were
found
for
the
samples
by
doses
of
10
-3
and
10
-4
mg/ml
ascorbic
acid
in
4
hours.
Both
samples
showed
greater
changes
for
mentioned
doses
in
one
week.
This
indicated
the
impact
of
ascorbic
acid
and
longer
time
of
treatment
on
the
strength
of
cornea
and
the
reduction
of
distance
between
collagen
fibrils
(consequently
the
increase
of
cross-linking).
The
dose
of
10
-3
mg/ml
ascorbic
acid
had
a
greater
effect
in
comparison
with
10
-4
mg/ml
ascorbic
acid
in
one
week.
Collagen
cross-linking
leads
to
new
covalent
bonds
between
the
collagen
strings.
These
bonds
affect
the
biomechanical
strength
and
stiffness
of
cornea.
Furthermore,
they
also
change
the
corneal
refractive
index
at
multiple
locations
in
cornea
and
reduce
its
adverse
effects
on
vision.
The
gradual
increase
of
visual
acuity
implies
the
efficacy
of
collagen
cross-linking
in
cornea
and
its
modifying
factors.
Wollensak
et
al.
(24)
studied
the
effects
of
riboflavin
and
UVA
rays
on
23
eyes
with
advanced
keratoconus.
They
showed
the
best
corrected
vision
was
raised
by
1.65
±
1.5
lines
in
vision
chart
for
65%
of
patients
(24).
Sander
et
al.
(25)
also
conducted
a
study
on
the
corneal
collagen
cross-linking
in
60
eyes
with
advanced
keratoconus.
They
concluded
that
the
best
corrected
vision
was
increased
about
2.04
±
1.4
lines
in
eye
chart
(25).
Caporossi
et
al.
(26)
reported
the
increase
of
6
3.6
and
1.66
lines
of
eye
chart
in
corrected
and
uncorrected
visual
acuity
for
10
patients
at
6
months
after
treatment,
respectively
(26).
Pinelli
(27)
showed
that
uncorrected
and
corrected
visual
acuity
has
been
increased
by
2
and
1.8
lines
in
the
eye
chart
at
nine
months
after
collagen
cross-linking
on
10
patients
with
advanced
keratoconus
(27).
Raiskup
et
al.
(28)
studied
272
patients
with
advanced
keratoconus.
They
showed
that
the
best
corrected
vision
in
53
%
of
patients
was
increased
more
than
one
line
in
the
eye
chart
at
the
first
year
after
collagen
cross-linking
while
it
did
not
change
for
20
%
of
patients
(28).
Our
results
showed
that
ascorbic
acid
with
different
doses
and
at
different
times
was
very
less
destructive
at
keratocyte
in
comparison
with
optisol.
These
were
inconsistent
with
cell
cultures
derived
from
keratocyte
of
pigs,
the
damage
threshold
for
UVA
ray
in
combination
with
riboflavin0.0025
was
determined
0.4
mw/cm2
which
was
10
times
lower
than
the
threshold
of
UVA
ray
alone
(29).
In
another
study
on
keratocytes
in
pigs,
Wollensaket
al.
(30)
showed
that
the
threshold
of
cellular
damage
of
keratocytes
were
0.5
mw/cm2
and
5
mw/cm2
for
a
combination
of
UVA
ray
and
riboflavin
0.1%
and
UVA
ray,
respectively
(30).
The
reduction
in
keratocytes
for
the
depth
of
270-350
micron
inside
stroma
were
observed
by
clinical
investigations
with
the
help
of
confocal
microscopy
on
10
corneal
cross-linking.
However,
it
was
completely
restored
by
the
adjacent
cell
mobilization
after
6
months
(26,
31).
The
low
number
of
samples
and
different
measurements
of
the
distance
between
collagen
fibrils
were
the
weaknesses
of
our
study.
The
novelty
of
this
work
was
to
study
the
effects
of
ascorbic
acid
on
the
increase
of
corneal
collagen
cross-linking.
The
purpose
of
the
current
study
was
to
determine
the
effects
of
ascorbic
acid
on
corneal
collagen
cross-linking
in
keratoconus
in
vitro.
Corneal
collagen
cross-linking
is
explained
as
the
most
promising
novelty
in
the
treatment
of
progressive
keratoconus
in
recent
years.
We
found
statistically
significant
increasing
in
the
resistance
of
stromal
collagen.
Results
showed
that
ascorbic
acid
can
strengthen
the
cornea
and
decrease
the
distance
between
collagen
fibrils
(consequently
increase
cross-linking).
Therefore,
the
efficacy
of
ascorbic
acid
is
observed
by
more
recovery
through
increasing
its
doses
and
passing
time.
It
is
noteworthy
that
this
study
can
be
a
useful
preliminary
to
future
researches.
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