Experimental Eye Research 165 (2017) 1e6

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Experimental Eye Research

journal homepage: www.elsevier.com/locate/yexer

Riboflavin and ultraviolet A irradiation for the prevention of

progressive myopia in a guinea pig model
Xiaoxia Li, Miaoqin Wu*, Luyi Zhang, Hui Liu, Lan Zhang, Jinjing He
Department of Ophthalmology, Zhejiang Provincial People's Hospital, People's Hospital of Hangzhou Medical College, Hangzhou, Zhejiang, China

a r t i c l e i n f o a b s t r a c t

Article history: In this study, we evaluated the effect of oral administration of riboflavin combined with whole-body
Received 26 September 2016 ultraviolet A (UVA) irradiation on the biochemical and biomechanical properties of sclera in a guinea
Received in revised form pig model to control the progression of myopia. Experimental groups were administered 0.1% riboflavin
27 August 2017
solution with or without vitamin C by gavage from 3 days before myopic modeling and during the
Accepted in revised form 28 August 2017
modeling process. Guinea pigs underwent 30 min of whole-body UVA irradiation after each gavage for 2
Available online 31 August 2017
weeks. For control groups, guinea pigs were administered vitamin C and underwent either whole-body
UVA irradiation without 0.1% riboflavin solution or whole-body fluorescent lamp irradiation with or
Keywords:
Myopia
without 0.1% riboflavin solution. Resultantly, myopia models were established with an increased axial
Riboflavin length and myopic diopter. Compared with myopic eyes in the control groups, the net increase in axial
Sclera length, diopter and strain assessment decreased significantly, and the net decrease in sclera thickness,
UVA ultimate load, and stress assessment decreased significantly in experimental groups. MMP-2 expression
showed a lower net increase, while TIMP-2 expression showed a lower net decrease. In addition, hy-
perplasia of scleral fibroblasts was more active in myopic eyes of experimental groups. Overall, our re-
sults showed that oral administration of riboflavin with whole-body UVA irradiation could increase the
strength and stiffness of sclera by altering the biochemical and biomechanical properties, and decreases
in axial elongation and myopic diopter are greater in the guinea pig myopic model.
© 2017 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction that the sclera behaves as a “metabolic disorder” (McBrien and

Myopia is a significant public health problem with an increasing
prevalence, and it is the leading cause of correctable visual
impairment in children and adults in the developed world and one
of the main causes of blindness in developing countries (Robaei
et al., 2006; Lu et al., 2009; Vitale et al., 2008; Resnikoff et al.,
2008). In China, there is a high prevalence of myopia, with an
average morbidity of more than 75% in young adults aged 15e25
years (Holden et al., 2014).
Myopia, particularly high myopia, is associated with major
ophthalmic diseases that can eventually lead to blindness, such as
exudative myopic macular degeneration, rhegmatogenous retinal
detachment, myopic retinopathy, and glaucoma. The mechanism of
myopia is not completely understood, but many researchers believe
* Corresponding author. Department of Ophthalmology, Zhejiang Provincial
People's Hospital, People's Hospital of Hangzhou Medical College, 158 Shangtang
Road, Hangzhou, Zhejiang 310014, China.
E-mail address: eyewmq@126.com (M. Wu).
Gentle, 2003). Various inborn and postnatal factors cause the
sclera to be thin and expand under eye pressure, leading to eyeball
deformations such as localized ectasia of the posterior sclera, which
results in myopia. Animal studies of experimental myopia have
shown that scleral tissue from myopic eyes decreases mechanical
stiffness and increases creep compliance compared with scleral
tissue from normal eyes (Phillips et al., 2000). The concept of
enhancing the strength and stiffness of the sclera to stabilize the
normal morphology of ocular tissue has attracted significant
research interest in recent years.
Wollensak and Iomdina reported stressestrain measurements
of a sector of the equatorial sclera of Chinchilla rabbit eyes, which
was treated in vivo with ultraviolet A (UVA) irradiation and ribo-
flavin drops, and proposed that scleral collagen cross-linking
induced by UVA and riboflavin may increase biomechanical
strength and scleral rigidity (Wollensak and Iomdina, 2009).
Another study investigated the in vitro ultrastructural effects of a
collagen cross-linking treatment with riboflavin instilled into the
strips prior to UVA irradiation in human corneo-scleral collagen

http://dx.doi.org/10.1016/j.exer.2017.08.019
0014-4835/© 2017 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
2 X. Li et al. / Experimental Eye Research 165 (2017) 1e6
fibrils and found that this method causes structural changes in the
collagen fibril network of the sclera (Choi et al., 2013). Riboflavin-
sensitized photoreaction generates free radicals and reactive oxy-
gen species, such as singlet oxygen, superoxide, or superoxide
anion radicals to induce cross-linking of collagen under UVA
(Wollensak and Spoerl, 2004; Wollensak et al., 2005). Scleral
collagen cross-linking by riboflavin/UVA is a novel technique to
enhance scleral mechanical strength, and may provide an ideal
approach for strengthening scleral tissue, thereby suppressing
myopic progression. However, these experiments using riboflavin
and UVA require a surgical operation to expose the sclera with a
limited irradiation area. At this time, further studies are required
for this intervention to be clinically applicable. Thus, we evaluated
the effects of whole-body UVA irradiation plus oral administration
of riboflavin on the biochemical and biomechanical properties of
sclera in myopic guinea pigs to develop a unique, non-invasive, and
practical therapy to control the progression of myopia. This is the
first report to investigate the effect of whole-body UVA irradiation
and riboflavin without surgical exposure of the sclera in myopic
eyes.
2. Materials and methods
2.1. Animals and treatments
All animal experiments conformed to “Regulations of Animal
Experiments in Zhejiang Province” and were approved by Zhejiang
Chinese Medical University. A total of 30 guinea pigs (all females,
four-weeks old) were treated with a
10.0 D concave lens on the
right eye for two weeks to establish the myopic model. The concave
lens was a
10.00 D poly(methyl methacrylate) (PMMA) lens
(Shanghai Freshkon Contact Lenses Co., Ltd., China) with a diameter
of 11.00 mm and inner arc curvature of 9.00 mm (Fig. 1). All guinea
pigs were randomly divided into five groups (n ¼ 6 in each):
group A (vitamin C þ whole-body fluorescent lamp irradiation,
control group), group B (vitamin C þ whole-body UVA irradiation,
control group), group C (vitamin C þ riboflavin þ whole-body
fluorescent lamp irradiation, control group), group D (vitamin
C þ riboflavin þ whole-body UVA irradiation, experimental group),
and group E (riboflavin þ whole-body UVA irradiation, experi-
mental group). As mentioned above, all guinea pigs were fed a basal
diet and 100 mg of vitamin C or 0.4 mg riboflavin in 0.1% solution by
gavage three times per day from three days before the beginning of
Fig. 1. Guinea pig models of lens-induced myopia. A: A single-use Murphy dropper
was pruned to construct a frame and the concave lens was agglutinated to the self-
made spectacle. B: The self-made spectacle was sutured and fixed to the right eye of
the guinea pig.
establishing myopia models to two weeks after the modeling.
During the modeling period, animals underwent fluorescent lamp
irradiation (30 cm from above) or UVA irradiation (370 ± 5 nm)
using a UVA device (Changjiang Medical Devices Co., Ltd, China)
with an irradiance of 3.0 mW/cm2 at a distance of 30 cm for whole-
body irradiation. For fluorescent lamp irradiation, the irradiation
started after each gavage and the total daily irradiation time was
8 h; for UVA irradiation, the irradiation started after each gavage
and lasted for 30 min, and the total daily irradiation time was 1.5 h.
The right eye of each animal served as the lens-induced eye and the
contralateral left eye served as the control eye.
2.2. Diopter and ocular axial length measurements
After general anesthesia by injecting ketamine hydrochloride
into the thigh muscles, the axial length was measured with A-Node
Ultrasonic Diagnostic Equipment (Meda Co., Ltd, China). Retinos-
copy optometry (Fizz Kang Visual Light Company, China) was used
to examine the diopter after cycloplegic pre- and post-experiments.
2.3. Biomechanical measurements
Guinea pigs were killed after two weeks and eyes were put into
preservation solution and cryopreserved in liquid nitrogen after
enucleation, after which the eyes were rewarmed rapidly in a
37e38  C water bath. Stressestrain tests were performed for sclera
specimens using a microcomputer-controlled biomaterial tester
(Instron Test Equipment Trade, Ltd, USA). The 15 mm  4 mm
scleral strips were clamped horizontally with a stress level of
0.005e0.04 N, and the strain was increased linearly at a velocity of
5 mm/min to test the tensile strength until the specimen broke. The
ultimate load, ultimate stress, and ultimate strain were recorded
using the microcomputer-controlled tester over time for further
analysis.
2.4. Posterior sclera thickness
After removing the vitreous, retina, and surrounding connective
tissue, scleral tissue (diameter, 6 mm) taken from the posterior
sclera was immediately immersed in 4% paraformaldehyde to be
fixed for 24 h, after which the sample was dehydrated and paraffin-
embedded to make a 4-mm section of the sclera; thin 4-mm sections
were stained with hematoxylin and eosin and periodic acid-Schiff
(PAS). A Zeiss light microscope (Axiomat, Zeiss, Oberkochen, Ger-
many) was used to measure the specimens (scleral tissue thickness
in the central region) at various magnifications.
2.5. Western blotting
Tissue homogenate was produced from 3 mm  4 mm sections
of posterior scleral tissue (100 mg) with 0.5e1 mL of cold lysis
buffer, after which it was centrifuged at 4  C and 10,000 rpm for
5 min. The protein concentrations were determined using bovine
serum albumin (BSA) as standards based on the Bradford assay
(Bio-Rad, BioTek Instruments, Inc. USA). The protein solution was
then separated by 10% polyacrylamide gel electrophoresis with 5%
stacking gels with sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE). The separated proteins were trans-
ferred onto nitrocellulose membranes, after which the membranes
were washed in Tris-buffered saline with 0.1% Tween-20 (TBS-T)
and blocked in plates with diluted matrix metalloproteinase-2
(MMP-2) antibody BA0569 (Wuhan Boster Biological Technology,
LTD., China) or tissue inhibitor of MMP-2 (TIMP-2) antibody BA0576
(Wuhan Boster Biological Technology, LTD., China) for 10e12 h at
4  C. Membranes were then washed three times in TBS-T and
 X. Li et al. / Experimental Eye Research 165 (2017) 1e6
incubated with secondary antibodies for 1 h at 26  C, after which
they were washed another three times in TBS-T. Protein blots on the
membranes were then enhanced by chemiluminescence (ECL
Detection Kit, Beyotime, China) and exposed using the FluorChem E
System. Films were scanned into digital images and the primary
densities of the band of MMP-2, TIMP-2, and glyceraldehyde-3-
phosphate dehydrogenase (GAPDH) were measured with Quan-
tity One (Bio-Rad).
2.6. Transmission electron microscopy
A small mark was made at the temporal corneal limbus using an
indelible ink marker pen to allow for orientation of the eye cup
after enucleation. Eyes were immersed in 0.05 M cacodylic acid
sodium and 2.5% glutaraldehyde with phosphate buffer saline (pH
7.4) for 2 h. After this period, the cornea and lens were dissected,
leaving the mark in the limbal region. A cornea trephine (diameter,
6 mm) was then used to punch out a posterior tissue button
(containing retina, choroid, and sclera). Two 2 mm  1 mm strips of
sclera tissue were excised from the tissue button near the nasal
region, 1 mm from the optic nerve with razor blades. The strips
were immersed in 0.05 M cacodylic acid sodium and 2.5% glutar-
aldehyde for 24 h. Tissue samples were post-fixed in 1% osmium
tetroxide with phosphate buffer saline (pH 7.4) for 1 h at 4  C,
stained with 1% uranyl acetate with double distilled water for 2 h,
and then rinsed and dehydrated in graded acetone before embed-
ding in Araldite. Scleral samples were analyzed using transmission
electron microscopy.
2.7. Statistical analyses
Statistical analysis with a significance level of p < 0.05 was
performed using Tukey's multiple comparisons test with SPSS
software (v17.0, SPSS Inc, USA). Data are presented as
mean ± standard deviation (SD).
3. Results
3.1. Establishment of myopia models
After two weeks, we compared pre-test and post-test parame-
ters of ocular axial length and diopter. The results showed that all
data in the lens-induced eye had significantly longer axial lengths
and higher myopic diopter compared with data from the pre-test,
which indicated that the myopic models were established
successfully.
As shown in Fig. 2, in control groups (A, B, C), mean axial length
in the lens-induced eye was 7.37 ± 0.25 mm (A), 7.23 ± 0.19 mm (B),
and 7.27 ± 0.24 mm (C) before establishing the myopic model, and
8.31 ± 0.09 mm, 8.25 ± 0.11 mm, and 8.11 ± 0.17 mm 14 days later,
respectively. In experimental groups (D, E), which underwent oral
administration of riboflavin and whole-body UVA irradiation, mean
axial length in the lens-induced eye were 7.09 ± 0.18 mm (D) and
7.19 ± 0.11 mm (E) at day 0 and 7.94 ± 0.10 mm and 7.99 ± 0.09 mm
14 days later, respectively. The measurement of the axial lengths
revealed a lower net increase in myopic eyes of experimental
groups (D, E) compared with myopic eyes in control groups (A, B, C)
(Fig. 2A). In the present study, significant differences were observed
between the control and experimental groups (A vs. D, A vs. E, B vs.
D, B vs. E, all p < 0.01). However, no significant difference (p > 0.05)
was observed between group C and experimental groups (D, E).
In control groups (A, B, C), mean diopter in the lens-induced eye
measured 3.37 ± 0.29 D (A), 3.40 ± 0.28 D (B), and 3.51 ± 0.29 D (C)
at day 0 and
2.21 ± 0.25 D,
2.26 ± 0.26 D, and
2.03 ± 0.10 D 14
days later, respectively. In experimental groups (D, E), mean diopter
3
in the lens-induced eye measured 3.33 ± 0.17 D (D) and 3.27 ± 0.16
D (E) at day 0 and
1.47 ± 0.13 D and
1.53 ± 0.22 D 14 days later,
respectively. A comparison of the mean changes in diopter of the
right eyes between control groups (A, B, C) and experimental
groups (D, E) yielded a lesser net increase for myopic eyes in the
experimental groups (D, E) (A vs. D, A vs. E, B vs. D, B vs. E, C vs. D, C
vs. E, all p < 0.01) (Fig. 2B). There was no significant difference in the
axial length and diopter (p > 0.05) between groups D and E. These
results demonstrated that our intervention method may suppress
the development of myopia.
3.2. Posterior scleral thickness and biomechanical measurements
The scleral thickness of the control eye was thicker than the
lens-induced eye in all groups, as indicated in Fig. 2C. In the
experimental groups (D, E), mean thickness of myopic eyes showed
a significantly lower net decrease (A vs. D, A vs. E, B vs. D, B vs. E, C
vs. D, C vs. E, all p < 0.01) compared with myopic eyes in control
groups (A, B, C). Although no significant difference was observed
between experimental groups (D, E), our results indicated that
systematic whole-body UVA irradiation with oral administration of
riboflavin could thicken the sclera in myopic eyes.
Compared with control eyes, the ultimate load and stress
assessment were lower and the strain assessment was higher for
myopic eyes in each group. Moreover, the ultimate load and stress
assessment for myopic eyes in the experimental groups were
significantly higher and strain assessment for myopic eyes in the
experimental groups was significantly lower than myopic eyes in
control groups (Fig. 2DeF). Fig. 2D showed that after 14 days of
myopic modeling, myopic eyes in experimental groups (D, E) had
higher values of ultimate load than myopic eyes in control groups
(A, B, C) (Fig. 2D, A vs. D, A vs. E, B vs. D, B vs. E, C vs. D, C vs. E, all
p < 0.01); Fig. 2E showed that after 14 days of myopic modeling,
myopic eyes in experimental group D had higher values of stress
assessment than myopic eyes in the control groups (A, B) (Fig. 2E, A
vs. D, B vs. D, all p < 0.01); Fig. 2F showed that after 14 days of
myopic modeling, myopic eyes in experimental groups (D, E) had
lower values of strain assessment than myopic eyes in control
groups (A, B) (Fig. 2F, A vs. D, B vs. D, all p < 0.01; B vs. E, p < 0.05).
These results indicated that sclera in high myopic eyes was more
flexible and its load bearing capacity was lower than emmetropia,
but whole-body UVA irradiation with oral administration of ribo-
flavin could increase the biomechanical characteristics of sclera in
myopic eyes.
3.3. Protein expression level of MMP-2 and tissue TIMP-2
MMP-2 and TIMP-2 play key roles in the development of
myopia. Visual signals may modulate gene expression of selected
MMPs and TIMPs to control scleral remodeling, the mechanical
properties of the sclera, axial elongation, and the refractive state
(Siegwart and Norton, 2005). It is believed that the increased
expression of MMP-2 and the decreased expression of TIMP-2
promote myopia.
The results shown in Fig. 3 demonstrate that whole-body UVA
irradiation plus oral administration of riboflavin regulates the
expression of MMP-2 and TIMP-2. In the control groups (A, B, C),
myopic treatment significantly increased the expression of MMP-2
(Fig. 3B) and significantly decreased the expression of TIMP-2
(Fig. 3C), whereas in the experimental groups (D, E), myopic
treatment did not significantly increase the expression of MMP-2
(Fig. 3B) and did not significantly decrease the expression of
TIMP-2 (Fig. 3C), but actually increased the expression of TIMP-2.
Overall, our results demonstrated that whole-body UVA irradia-
tion plus oral administration of riboflavin could suppress myopia
4 X. Li et al. / Experimental Eye Research 165 (2017) 1e6

Fig. 2. Ocular axial length, diopter, posterior scleral thickness, ultimate load, stress assessment, and strain assessment data. A: Significant differences in axial length were observed
between groups in the lens-induced eye at 14 days (**D p < 0.01 vs. A, B, **E p < 0.01 vs. A, B). B: Comparison of the mean diopter of lens-induced eyes at 14 days yielded a
significantly lower value (**D p < 0.01 vs. A, B, C; **E p < 0.01 vs. A, B, C). C: Scleral thickness in the lens-induced eye: **D p < 0.01 vs. A, B, C; **E p < 0.01 vs. A, B, C. D: Ultimate load in
the lens-induced eye: **D p < 0.01 vs. A, B, C; **E p < 0.01 vs. A, B, C. E: Ultimate stress in the lens-induced eye: **D p < 0.01 vs. A, B. F: Ultimate strain in the lens-induced eye: **D p <
0.01 vs. A, B; *E p < 0.05 vs. B.

Fig. 3. Western blot analysis of MMP-2 and TIMP-2. (GAPDH: glyceraldehyde-3-phosphate dehydrogenase). A: Representative picture of Western blot analysis (WB). B: Statistical
summary of the expression of MMP-2 in WB. C: Statistical summary of the expression of TIMP-2 in WB. **: p < 0.01 vs. control.

progression.
3.4. Transmission electron microscopy of scleral tissue
Transmission electron microscopy was used to detect the hy-
perplasia of fibroblasts in scleral tissue 14 days after treatment.
Compared with control groups (A, B, C), the density of scleral fi-
broblasts increased significantly in myopic eyes in the experimental
groups (D, E), and more fibroblasts were present in myopic eyes in
group D and E (Fig. 4). In total, whole-body UVA irradiation plus
oral administration of riboflavin could enhance the hyperplasia of
fibroblasts in scleral tissue.
 X. Li et al. / Experimental Eye Research 165 (2017) 1e6
Fig. 4. Photomicrograph of the sclera in different groups demonstrating the hyperplasia of fibroblasts at 14 days after myopic treatment. Arrows indicate the aggregation and
cellular bundles of fibroblasts in the sclera.
4. Discussion
Myopia is often accompanied by decreased scleral collagen
content, changes in collagen fiber diameter and distribution, and
decreased scleral thickness (McBrien et al., 2001). Riboflavin and
UVA treatment, as well as other chemical cross-linking methods
such as glutaraldehyde injection (Charulatha and Rajaram, 2003),
are rarely used to control myopia due to their potential debatable
roles in the sclera. The applications of these methods are limited
due to the available area of irradiation, the requirement of surgical
exposure of the sclera (Wollensak and Iomdina, 2009), and the
potential cytotoxic risk to the corneal endothelium, lens, and retina
at an inappropriate dosage (Wollensak et al. 2003, 2005; Zhao et al.,
2013). Our data suggest that systematic whole-body UVA irradia-
tion plus oral administration of riboflavin can effectively improve
the structural, biochemical, and biomechanical characteristics of
sclera, delaying myopic development in a guinea pig myopic model.
Indeed, myopia-induced decreases in the diameter of collagen
fibrils and collagen fiber bundles result in elongation and localized
ectasia of the sclera (Rada et al., 2006). Moreover, elongation of the
eyes is determined mechanistically by the biochemical and
biomechanical properties of the sclera (Rada et al., 2000). Common
approaches to change the biochemical and biomechanical proper-
ties of the sclera include surgical exposure of the sclera, which is
invasive and painful. In this study, we attempted to identify a novel
approach to change the biochemical and biomechanical properties
of the sclera. We showed that the oral administration of riboflavin
combined with whole-body UVA irradiation without surgical
exposure of the sclera and direct UVA irradiation on the sclera
could suppress axial length elongation and myopic diopter incer-
asing in a guinea pig myopic model.
Pathological changes in the sclera with progressive scleral
thinning and a decreased biomechanical extensibility are impor-
tant features and common causes of severe myopia (McBrien and
Gentle, 2003; Rada et al., 2006). Choi et al. suggested that the
collagen cross-linking technique induced by riboflavin and UVA
leads to increases in the density, area, diameter, and thickness of
human scleral tissues (Choi et al., 2013). In the present study, the
experimental groups with oral riboflavin and whole-body UVA
irradiation showed a lower net decrease in the development of
myopia, especially in scleral thickness and ultimate load, suggest-
ing that our method may be associated with scleral collagen cross-
linking in the myopic eye.
During the development of myopia, the sclera undergoes active
remodeling that involves the regulation of MMPs and TIMPs (Kim
et al., 2001; Shelton and Rada, 2007). Therefore, the expression
variations of these two proteins among the groups were evaluated
in our myopic model. Increased scleral MMP-2 expression in form-
deprivation myopia has been observed in guinea pigs at the protein
level (Wang et al., 2010). Meanwhile, MMP-2 can be controlled by
numerous factors including down-regulation of the expression of
5
specific proteins, such as TIMPs (Siegwart and Norton, 2002).
Biomechanical scleral changes in myopia include of a net loss of the
matrix induced by increases in levels of both latent and active
MMP-2 (Rada et al., 2006; Guggenheim and McBrien, 1996). These
results were confirmed in the present study, showing that the
expression of MMP-2 increased and the expression of TIMP-2
decreased in lens-induced eyes. Interestingly, myopic eyes in the
experimental groups showed a lower net increase in MMP-2 and a
lower net decrease of TIMP-2, suggesting that our method does not
strongly affect the sclera. However, further studies are required to
explore the underlying mechanisms.
Based on electron microscopy, the sclera consists of fibroblasts
and collagen bundles, which can increase strength through changes
in the biomechanical properties of the sclera. In myopic eyes in our
control groups (A, B, C), the diameter of the posterior sclera fibro-
blasts was thinner compared with that of myopic eyes in experi-
mental groups (D, E), and there was vacuolar degeneration in the
fibroblasts. A previous report showed that the major differences
were in morphology and in the diameter of fibroblasts in the pos-
terior sclera between normal and myopic eyes (Liu et al., 1986),
which agrees with our results.
According to the above reports, riboflavin, no systemic side ef-
fects, can increase the absorption of UVA to limit the damage of
UVA to ocular tissues under the appropriate doses during collagen
cross-linking (Zhao et al., 2013). However, no reports have provided
the exact dose of orally administered riboflavin, which is important
to ensure that the concentration in the sclera is sufficient to have a
therapeutic effect.
The effects of UVA on the retina were a concern during this
study. Thus, the parameters of UVA irradiation were carefully
selected. Wollensak and Iomdina (2009) confirmed that there were
no side effects on the retina or retinal pigment epithelium using
UVA under 370 nm and 3 mW/cm2. Moreover, UVA was applied for
collagen cross-linking at 370 nm, where riboflavin absorption
peaks and there is no evidence of tissue damage in this band (Zhao
et al., 2013). Therefore, in this study, UVA at 370 nm and 3 mW/cm2
were selected and UVA irradiation was applied using a whole-body
irradiation approach.
Vitamin C, unlike other antioxidants, does not absorb radiation
in the UVA range (Ou-Yang et al., 2004) and can slow the absorption
and degradation of riboflavin (Lee et al., 1998). Moreover, vitamin C
can counteract DNA damage and mutagenesis induced by the UVA
and riboflavin (Besaratinia et al., 2007). Therefore, the possibility of
vitamin C to interfere with the optical effects of UVA and riboflavin
could be ruled out. The protective effects of vitamin C observed in
this study may be ascribed to its antioxidant and free radical
scavenging capacities (Catani et al., 2005).
In summary, we present the first in vivo study on decreasing
axial length elongation and limiting increases in myopic diopter in
a guinea pig myopic model using non-invasive oral administration
of riboflavin combined with whole-body UVA irradiation. The
6 X. Li et al. / Experimental Eye Research 165 (2017) 1e6
above combinative intervention led to significant changes in pro-
tein levels of MMP-2 and TIMP (related to scleral remodeling) and
biomechanical rigidity of the sclera in the myopic model mentioned
above. In our study, we found no significant difference in the axial
length, stress assessment and strain assessment between group C
with experimental groups (D, E). It is known that the wavelength of
fluorescent lamps range from 400 to 700 nm (Pasquale et al., 2017;
Karin et al., 2017). Within this field, some wavelengths of light may
have an effect, similar to UVA (370 ± 5 nm), to delay myopic
development with the oral administration of riboflavin. Further
systemic studies are required to determine the underlying mech-
anisms, address the potential toxicity (such as impairment of the
retina), and develop protective solutions (such as protection of the
retina).
Conflict of interest
The authors in this article have no potential conflicts of interest.
Disclosure
Contributions of authors: Designing the study (X.X.L., M.Q.W.);
conducting the study (H.L., L.Z.); collecting and managing the data
(X.X.L., H.L., L.Z.); analyzing and interpreting the data (X.X.L., L.Y.Z.,
J.J.H.); preparing, reviewing, and approving the manuscript (X.X.L.,
M.Q.W.).
Acknowledgments
We thank 100Biotech Co. Ltd for assistance of performing pretest
and affording the labs. This Project was supported by Zhejiang
Province Science and Technology Department (2014C03042-1).
References
Besaratinia, A., Kim, S.I., Bates, S.E., Pfeifer, G.P., 2007. Riboflavin activated by ul-
traviolet A1 irradiation induces oxidative DNA damage-mediated mutations
inhibited by vitamin C. Proc. Natl. Acad. Sci. U. S. A. 104, 5953e5958.
Catani, M.V., Savini, I., Rossi, A., Melino, G., Avigliano, L., 2005. Biological role of
vitamin C in keratinocytes. Nutr. Rev. 63, 81e90.
Charulatha, V., Rajaram, A., 2003. Influence of different crosslinking treatments on
the physical properties of collagen membranes. Biomaterials 24, 759e767.
Choi, S., Lee, S.C., Lee, H.J., Cheong, Y., Jung, G.B., Jin, K.H., Park, H.K., 2013. Structural
response of human corneal and scleral tissues to collagen cross-linking treat-
ment with riboflavin and ultraviolet A light. Lasers. Med. Sci. 28, 1289e1296.
Guggenheim, J.A., McBrien, N.A., 1996. Form-deprivation myopia induces activation
of scleral matrix metalloproteinase-2 in tree shrew. Invest. Ophthalmol. Vis. Sci.
37, 1380e1395.
Holden, B., Sankaridurg, P., Smith, E., Aller, T., Jong, M., He, M., 2014. Myopia, an
underrated global challenge to vision: where the current data takes us on
myopia control. Eye (Lond) 28, 142e146.
Karin, K., Takayuki, T., Mark, A.S., 2017. Performance of arabidopsis thaliana under
different light qualities: comparison of light-emitting diodes to fluorescent
lamp. Funct. plant Biol. 1445e4408.
Kim, J.W., Lindsey, J.D., Wang, N., Weinreb, R.N., 2001. Increased human scleral
permeability with prostaglandin exposure. Invest. Ophthalmol. Vis. Sci. 42,
1514e1521.
Lee, K.H., Jung, M.Y., Kim, S.Y., 1998. Effects of ascorbic acid on the light-induced
riboflavin degradation and color changes in milks. J. Agric. Food. Chem. 46,
407e410.
Liu, K.R., Chen, M.S., Ko, L.S., 1986. Electron microscopic studies of the sclera
collagen fiber in excessively high myopia. Taiwan. yi. xue. hui. za. Zhi 85,
1032e1038.
Lu, Q., Zheng, Y., Sun, B., Cui, T., Congdon, N., Hu, A., Chen, J., Shi, J., 2009.
A population-based study of visual impairment among pre-school children in
Beijing: the Beijing study of visual impairment in children. Am. J. Ophthalmol.
147, 1075e1081.
McBrien, N.A., Cornell, L.M., Gentle, A., 2001. Structural and ultrastructural changes
to the sclera in a mammalian model of high myopia. Invest. Ophthalmol. Vis.
Sci. 42, 2179e2187.
McBrien, N.A., Gentle, A., 2003. Role of the sclera in the development and patho-
logical complications of myopia. Prog. Retin. Eye. Res. 22, 307e338.
Ou-Yang, H., Stamatas, G., Saliou, C., Kollias, N., 2004. A chemiluminescence study of
UVA-induced oxidative stress in human skin in vivo. J. Invest. Dermatol 122,
1020e1029.
Pasquale, O., Luigi, G., Massimiliano, M., Stefano, O., 2017. The photoluminescence of
a fluorescent lamp: didactic experiments on the exponential decay. Phys. Educ.
52, 015011.
Phillips, J.R., Khalaj, M., McBrien, N.A., 2000. Induced myopia associated with
increased scleral creep in chick and tree shrew eyes. Invest. Ophthalmol. Vis.
Sci. 41, 2028e2034.
Rada, J.A., Achen, V.R., Penugonda, S., Schmidt, R.W., Mount, B.A., 2000. Proteo-
glycan composition in the human sclera during growth and aging. Invest.
Ophthalmol. Vis. Sci. 41, 1639e1648.
Rada, J.A., Shelton, S., Norton, T.T., 2006. The sclera and myopia. Exp. Eye. Res. 82,
185e200.
Resnikoff, S., Pascolini, D., Mariotti, S.P., Pokharel, G.P., 2008. Global magnitude of
visual impairment caused by uncorrected refractive errors in 2004. Bull. World.
Health. Organ 86, 63e70.
Robaei, D., Huynh, S.C., Kifley, A., Mitchell, P., 2006. Correctable and non-correctable
visual impairment in a population-based sample of 12-year-old Australian
children. Am. J. Ophthalmol. 142, 112e118.
Shelton, L., Rada, J.S., 2007. Effects of cyclic mechanical stretch on extracellular
matrix synthesis by human scleral fibroblasts. Exp. Eye. Res. 84, 314e322.
Siegwart Jr., J.T., Norton, T.T., 2002. The time course of changes in mRNA levels in
tree shrew sclera during induced myopia and recovery. Invest. Ophthalmol. Vis.
Sci. 43, 2067e2075.
Siegwart Jr., J.T., Norton, T.T., 2005. Selective regulation of MMP and TIMP mRNA
levels in tree shrew sclera during minus lens compensation and recovery.
Invest. Ophthalmol. Vis. Sci. 46, 3484e3492.
Vitale, S., Ellwein, L., Cotch, M.F., Ferris 3rd, F.L., Sperduto, R., 2008. Prevalence of
refractive error in the United States, 1999e2004. Arch. Ophthalmol. 126,
1111e1119.
Wang, S.R., Ye, J.J., Long, Q., 2010. Expressions of collagen I, matrix
metalloproeinases-2, and tissue inhibitor of matrix metalloproteinase-2 in the
posterior sclera of newborn Guinea pigs with negative lens-defocused myopia.
Zhongguo. Yi. Xue. Ke. Xue. Yuan. Xue. Bao 32, 55e59.
Wollensak, G., Iomdina, E., 2009. Long-term biomechanical properties of rabbit
sclera after collagen crosslinking using riboflavin and ultraviolet A (UVA). Acta.
Ophthalmol. 87, 193e198.
Wollensak, G., Iomdina, E., Dittert, D.D., Salamatina, O., Stoltenburg, G., 2005. Cross-
linking of scleral collagen in the rabbit using riboflavin and UVA. Acta. Oph-
thalmol. Scand. 83, 477e482.
Wollensak, G., Spoerl, E., 2004. Collagen crosslinking of human and porcine sclera.
J. Cataract. Refract. Surg. 30, 689e695.
Wollensak, G., Spoerl, E., Wilsch, M., Seiler, T., 2003. Endothelial cell damage after
riboflavin-ultraviolet-A treatment in the rabbit. J. Cataract. Refract. Surg. 29,
1786e1790.
Zhao, X., Wang, M.M., Zhang, F.J., 2013. Advanced research in the safety of UVA-
riboflavin cross-linking for strengthening corneal and scleral tissue. Zhong-
hua. Yan. Ke. Za. Zhi 49, 189e192.