Role of Corneal Collagen Cross-Linking in

Pseudophakic Bullous Keratopathy

A Clinicopathological Study
Ritu Arora, MD,1 Aditi Manudhane, MS,1 Ravindra Kumar Saran, MD,2 Jawaharlal Goyal, MD,1
Gaurav Goyal, MS,1 Deepa Gupta, MS1
Objective: To evaluate the clinical and histopathologic changes induced by collagen cross-linking (CXL) in
pseudophakic bullous keratopathy (PBK).
Design: Randomized, prospective, interventional study.
Participants: Twenty-four patients with PBK were included in the study.
Methods: Twenty-four patients with PBK underwent CXL followed by keratoplasty at 1 or 3 months. Twelve
patients underwent penetrating keratoplasty 1 month after CXL (group A) and the remaining 12 patients underwent penetrating keratoplasty 3 months after CXL (group B). The main outcome measures were assessed at 1
week and 1 month for all patients and at 3 months for 12 patients only. The corneal buttons underwent histopathologic and immunouorescence evaluation.
Main Outcome Measures: Visual acuity, ocular discomfort (tearing, redness, pain), corneal haze, central
corneal thickness, histopathologic evaluation, and immunouorescent microscopy.
Results: Mean visual acuity showed a signicant improvement after CXL, from 1.925!0.173 before surgery
to 1.75!0.296 at 1 month after surgery (P 0.010), but deteriorated to 1.81!0.23 at 3 months. Symptomatic
relief after CXL was at a maximum at 1 month, with a worsening trend at 3 months. Eighteen patients showed
a reduction in corneal haze 1 month after CXL. The effect was maintained in 9 of 12 patients at 3 months. The
mean central corneal thickness decreased signicantly from 846.46!88.741 to 781.0!98.788 mm at 1 month
(P<0.01) after CXL, but increased to 805.08!136.06 mm at 3 months. Immunouorescence microscopy revealed
anterior stromal compaction in 7 of 12 patients (58.3%) in group A and in 5 of 12 patients (41.6%) in group B.
Staining of keratocyte nuclei with 40 ,6-diaminido-2-phenylindole dihydrochloride (Molecular Probes, Carlsband,
CA) revealed a relative uniform distribution throughout the stroma.
Conclusions: Collagen cross-linking causes symptomatic relief and a decrease in central corneal thickness
and anterior stromal compaction in PBK. However, the effect decreases with time and depends on disease severity.
Financial Disclosure(s): The author(s) have no proprietary or commercial interest in any materials discussed
in this article. Ophthalmology 2013;120:2413-2418 2013 by the American Academy of Ophthalmology.
Corneal edema is caused by accumulation of uid in the
extracellular spaces between the stromal lamellae, which
primarily is the result of endothelial pump dysfunction.1
Alterations in corneal morphologic features in the form of
surface irregularity and increased interbrillar spacing
result in decreased visual acuity, pain, watering, and
conjunctival hyperemia. Pseudophakic bullous keratopathy
(PBK) is the leading cause of ocular morbidity in patients
who have undergone cataract surgery.2
Various management options for PBK include the use of
therapeutic bandage contact lens,3 topical hyperosmotic
agents,4 electrocautery to Bowmans membrane,5 anterior
stromal puncture,6 conjunctival aps,7 amniotic membrane
transplantation,8 and excimer laser phototherapeutic
keratectomy.9 All of these procedures bring about only
symptomatic relief. Corneal transplantation, however,
remains the denitive treatment for a large number of
patients with PBK.10
Corneal collagen cross-linking (CXL) is a new treatment
used to increase the biomechanical strength of corneal
! 2013 by the American Academy of Ophthalmology
Published by Elsevier Inc.

tissue. Corneal CXL aims at creating additional chemical

bonds inside the corneal stroma by means of a highly
localized photopolymerization while minimizing exposure
to the surrounding structures of the eye.11 It is being used
successfully
in
the
treatment
of
progressive
keratoconus,11,12 stromal melt and thinning disorders,13
postsurgical ectasia,14 and corneal ulcers.15,16 Corneal
CXL has been shown to inuence the swelling behavior of
corneal tissue. Wollensak et al17 showed the changes in the
hydration behavior of normal de-epithelialized porcine
corneas after CXL. Treated corneas were found to be more
transparent. Recent studies18e21 have shown the benecial
effect of CXL on clinical parameters like central corneal
thickness (CCT) and ocular discomfort in patients with
corneal edema.
Studies using animal22 and human23 models have
demonstrated massive keratocyte apoptosis in the anterior
stroma after CXL, with gradual repopulation by migration
of surrounding activated keratocytes into the treated area.
Using immunouorescence microscopy, Botts et al24,25
ISSN 0161-6420/13/$ - see front matter
http://dx.doi.org/10.1016/j.ophtha.2013.07.038

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Ophthalmology Volume 120, Number 12, December 2013

observed collagen ber organization, demonstrating the
cross-linking effect induced by CXL in corneas of patients
with corneal edema. However, the study included only
7 patients, and there was wide variability in the time gap
between CXL and immunouorescence microscopy. The
aim of our study was to evaluate the clinical and histopathologic changes after CXL in moderate to severe corneal
edema in 24 patients with PBK at 1 or 3 months after
surgery.

Methods
Twenty-four patients with corneal edema resulting from PBK of
more than 4 months duration and awaiting keratoplasty were
enrolled to undergo CXL followed by penetrating keratoplasty.
They were allocated randomly into 2 groups of 12 patients each
using computer-generated tables at the time of enrollment: 12
patients underwent penetrating keratoplasty 1 month after CXL
(group A), whereas the remaining 12 patients underwent penetrating keratoplasty 3 months after CXL (group B). All eligible
patients signed an informed consent form at the time of enrolment
in the study. The inclusion criteria for the study were between 30
and 70 years of age, endothelial decompensation lasting more than
4 months after cataract surgery, and CCT of more than 550 mm.
Patients with intraocular pressure of more than 22 mmHg, corneal
opacities, dry eye, or a history of photosensitivity were excluded
from the study.
The primary outcome of the study was the effect on CCT,
ocular discomfort, and histopathologic modications of corneal
stroma at 1 or 3 months after CXL. The research was approved by
the ethics committee of the institution and adhered to the tenets of
Declaration of Helsinki.
Before CXL treatment, patients were using hyperosmolar drops
(hyperosmotic sodium chloride 5% 4 times daily). All the participating patients underwent serial clinical examinations, including
visual acuity, grading of ocular discomfort, and pachymetry before
surgery and during subsequent visits after CXL at 1 week and 1
month. Only 12 patients in group B were followed up to 3 months
after CXL. At the time of keratoplasty, the corneal buttons were
processed appropriately and were submitted for detailed histopathologic analysis. All 24 samples were evaluated by a single
masked observer (R.K.S.).
The visual acuity of all patients was recorded at each visit using
Snellens chart and later was converted to the logarithm of the
minimum angle of resolution scale. The visual analog scale
(VAS)26 was used to quantify ocular discomfort, which included
excessive tearing, redness, and pain. The patients were asked to
mark on the line, from 1 to 10, the point that they believed
represented their perception of pain. The VAS score was
determined by measuring from the left hand end of the line to
the point marked by the patient. Zero stood for no pain, whereas
10 stood for unbearable pain. Corneal haze was categorized into
4 grades24 on slit-lamp examination: 1 corneal edema in the
posterior stroma; 2 microcystic epithelial edema plus corneal
edema in the posterior stroma; 3 epithelial erosions and bullae
formations; and 4 brosis of corneal surface.
Central corneal thickness was measured using ultrasound
pachymetry (Sonomed-PacScan plus; New York, NY) and
noncontact anterior segment optical coherence tomography (OCT;
RTvue-100 [Freemont, CA] with monochrome charge-coupled
device camera). Anterior segment OCT was found to record CCT
in severely edematous corneas where ultrasound pachymetry was
difcult. Central corneal thickness was measured before surgery in
all patients and at subsequent visits after CXL. Corneal CXL was

2414

performed according to the standard Dresden protocol.11

Penetrating keratoplasty was performed at 1 and 3 months.
Corneal buttons were sent for histopathologic analysis.

Histopathologic Analysis of Corneal Buttons Using

Immunouorescence Microscopy
After surgical removal, the host corneal buttons were embedded in
a tissue-freezing media at $70% C (snap freeze technique). Thin
sections (5 mm) were obtained on a cryostat at $21% C. The slides
were washed with phosphate-buffered saline and then were incubated with primary antibodies: anticollagen type I mouse antibody
(1:300; Calbiochem, Darmstadt, Germany) for 1 hour. Antibodies
to type 1 collagen were selected for immunouorescence because
90% of corneal collagen is type 1 collagen.27 The slides then were
washed with phosphate-buffered saline solution and were incubated for 30 minutes with antimouse immunoglobulin G secondary
antibody (1:300) conjugated with a chromogen Alexauor 488
(Molecular Probes, Carlsband, CA). The slides were washed
further and were stained with a nuclear stain 40 ,6-diaminido-2fenilindole dihydrochloride (DAPI; Molecular Probes) at
a 1:1000 dilution in phosphate-buffered saline for 5 minutes. The
DAPI staining was used to investigate the integrity and arrangement of keratocytes in stroma. The sections were mounted using
glycerine and were observed under an Olympus BX51 uorescence
microscope using a WB-2/WU-5 lter with 494-nm wavelength for
excitation and 518-nm wavelength for emission.
Images were obtained using Olympus DP71 camera coupled
with Image-Pro Plus 6.2 software (Media Cybernetics, Inc.,
Rockville, MD). Slides untreated with primary antibodies were
used as negative controls in both groups.

Statistical Analysis
Standard follow-up visits were made at 1 week, 1 month, and
3 months for the purpose of statistical analysis. Parameters were
compared between the 2 groups and within each group at each
follow-up visit with the preoperative ndings. Data were analyzed
using the paired t test, the nonparametric ManneWhitney U test,
and the Wilcoxon signed-rank test wherever appropriate. A P value
less than 0.05 was considered signicant.

Results
The mean visual acuity of patients in group A improved from
1.94!0.2 before surgery to 1.76!0.44 at 1 week and 1.88!0.22 at
1 month after CXL (Table 1). This change was not found to be
statistically signicant (P 0.47). The mean visual acuity of the
12 patients in group B improved from 1.91!0.15 before surgery
to 1.64!0.34 at 1 week (P 0.02), 1.62!0.31 at 1 month (P
0.006), and 1.81!0.23 at 3 months (P 0.083) after CXL.
Corneal CXL improved vision signicantly 1 month after the
procedure; however, this improvement was not sustained at 3
months (Table 2).
The mean VAS scores of patients in group A showed a statistically signicant decrease from 4.25!1.138 before surgery to
1.67!0.651 and 1.83!0.835 at 1 week and 1 month, respectively,
after CXL (Table 1). In the 12 patients followed up to 3 months
after CXL in group B, the mean VAS scores decreased from
5.25!1.357 before surgery to 2.08!1.084, 2.17!1.03, and
2.67!1.231 at 1 week, 1 month, and 3 months, respectively. The
VAS score showed a worsening trend to 2.67!1.231 for
12 patients at 3 months, but it was still signicantly lower than
the preoperative value (Table 2).

Arora et al

&

CXL in Pseudophakic Bullous Keratopathy

Table 1. Results of 12 Patients in Group A Followed up until 1 Month after Surgery

Outcome
Visual acuity
VAS
CCT (ASOCT)
CCT (UP; n 11)

Before Surgery

1 Week after Surgery

P Value

1 Month after Surgery

P Value

1.94!0.2
4.25!1.14
837.83!83.96
817.09!65.08

1.76!0.44
1.67!0.65
780.92!78.45
757.45!63.05

0.115
0.002
0.007
0.00

1.88!0.22
1.83!0.84
787.58!84.7
788.73!77.82

0.47
0.002
0.011
0.029

ASOCT anterior segment optical coherence tomography; CCT central corneal thickness; UP ultrasonic pachymeter; VAS visual analog scale.
Data are mean ! standard deviation unless otherwise indicated.

A trend of reduction in corneal haze was noted in group A and

group B after CXL. Overall, 18 patients showed a reduction in
corneal haze from 3 to 2 at 1 month. The effect was maintained
in 9 patients in group B at 3 months (Fig 1).
In group A, the mean CCT using anterior segment OCT was
837.83!83.96 mm before surgery, which decreased to
780.92!78.45 mm at 1 week and 787.58!84.69 mm at 1 month
after CXL (Table 1). This decrease in CCT was statistically
signicant at 1 week (P 0.007) and 1 month (P 0.011) after
CXL. In patients followed up to 3 months (group B) after CXL,
the mean CCT decreased from 855.08!96.20 mm before CXL to
767.00!132.57 mm at 1 week after CXL (P 0.077) and
774.42!114.62 mm at 1 month after CXL (P 0.013),
increasing to 805.08!136.06 mm at 3 months after CXL (P
0.084; Table 2).
In group A, the mean CCT using ultrasound pachymetry was
817.09!65.02, 757.45!63.05, and 788.73!77.81 mm before
surgery, 1 week after CXL, and 1 month after CXL, respectively
(Table 1). The data were recordable from 11 patients only. In
patients in group B, the mean CCT using ultrasound pachymetry
was 809.08!88.70 mm before CXL and reduced to
734.20!83.50 mm at 1 week after CXL (P 0.025) and
704.40!74.12 mm at 1 month after CXL (P 0.001); it
gradually increased to 732.30!79.76 mm at 3 months after CXL
(P 0.010). The data were recordable from 10 patients only
(Table 2). Ultrasound pachymetry was unable to record the CCT
in 1 patient in group A and in 2 patients in group B. These
patients had severe disease and had a high CCT that could not
be recorded with ultrasound pachymetry. A comparison of CCT
readings using anterior segment OCT and ultrasound pachymetry
using Pearsons correlation coefcient revealed a linear
relationship before CXL and at 1 month in group A and before
CXL and at 1 week and 1 month after CXL in group B.
The corneal buttons obtained from patients at the time of
keratoplasty at 1 month or 3 months after CXL were graded on
histopathologic examination as having mild, moderate, or
advanced disease. The severity of disease was based on interbrillar spacing, uid vacuolation, and irregularity in arrangement of

collagen bers. The sections were evaluated critically for stromal

compaction and for the pattern of distribution of stromal keratocytes. In group A, 4 samples showed histopathologically moderate
disease, whereas 8 showed advanced disease. In group A, 7 of 12
samples (58.6%) showed compaction of the anterior stroma. This
included all 4 patients with moderate disease (Fig 2A) and 3
patients with histopathologically advanced disease (Fig 2C). In
group B, 4 samples showed moderate disease, whereas 8 samples
showed advanced disease. Five of 12 samples (41.3%) showed
anterior stromal compaction. This included all 4 patients with
moderate disease (Fig 2E) and 1 patient with advanced disease
(Fig 2G).
Keratocyte nuclei stained with DAPI appeared as blue bodies
(Fig 2B, D, F, H). A relatively uniform distribution of keratocytes
was noted throughout the stroma in the samples of both groups.
The DAPI staining also demonstrated an intact epithelium in all
samples, suggesting that the epithelium had regrown in all cases
after CXL.

Discussion
Postsurgical corneal edema is one of the leading indications
for penetrating keratoplasty. Corneal transplantation alone
provides permanent visual recovery and relief of symptoms.
In developing countries with a shortage of donor corneas
and huge waiting lists of patients awaiting corneal transplantation, patients need to be provided with relief of
symptoms and, if possible, temporary improvement in
vision for the interim.
Corneal CXL is being advocated of late as a new tool in
the armamentarium for the temporary reduction in corneal
edema in patients with bullous keratopathy. It has been
found to improve corneal transparency, corneal thickness,
and ocular pain after surgery.21,22
Previous studies have reported signicant relief in
symptoms of ocular discomfort at up to 6 months, with

Table 2. Results of 12 Patients in Group B Followed up until 3 Months after Surgery

Outcome
Visual acuity
VAS
CCT (ASOCT)
CCT (UP)

Before Surgery

1 Week after Surgery

P Value

1 Month after Surgery

P Value

3 Months after Surgery

P Value

1.91!0.15
5.25!1.357
855.08!96.202
809.08!88.703

1.64!0.34
2.08!1.084
767.00!132.568
734.20!83.50

0.020
0.002
0.077
0.025

1.62 !0.31
2.17!1.03
774.42!114.62
704.40!74.123

0.006
0.002
0.013
0.001

1.81!0.23
2.67!1.231
805.08!136.064
732.30!79.762

0.083
0.003
0.084
0.010

ASOCT anterior segment optical coherence tomography; CCT central corneal thickness; UP ultrasonic pachymeter; VAS visual analog scale.
Data are mean ! standard deviation unless otherwise indicated.

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Ophthalmology Volume 120, Number 12, December 2013

Figure 1. Clinical photographs of a patient in group B: (A) before corneal collagen cross-linking (CXL), (B) 1 month after CXL, and (C) 3 months after CXL.

visual improvement at 1 month by reducing the formation of

microbullae and macrobullae.20,21 A similar trend of
improved VAS scores was seen in our study, the effect
being maximum at 1 month. Visual analog scale scores at 3
months, although greater than those at 1 month, were still
lower than the preoperative values. This symptomatic relief
probably resulted from CXL-induced stromal compaction
and reduced bullae formation.
Central corneal thickness has been reported20 to decrease
by a mean of 124 mm at 1 month, but at 6 months, the
reduction in CCT was only 37 mm. A recent study by
Gharaee et al21 evaluated the effect of CXL in PBK in 20
eyes over 6 months and did not nd any signicant fall in
CCT. Average CCT before CXL in the current study was
840 mm (24 patients), whereas in other studies, it has been
reported as 700 to 750 mm.20,28 Cases in the present
study, although more advanced, did show an average
decrease in CCT by 50 mm at 1 month, which increased at 3
months but was less than the preoperative values. Corneal
CXL proved to be effective even in advanced cases at 1
month, but the effect was not sustained up to 3 months after
CXL. The decrease in CCT could be attributed to the crosslinking effect causing compaction in the early period, but
the effect was not long lasting, probably because of
decreased riboavin diffusion across the highly edematous
corneas.
Corneal CXL improves the biomechanical stability of
corneal tissue by increasing the diameter of the collagen
bers29 and linking them in close association with each
other. The maximum effect of cross-linking is known to
be conned to the anterior stroma.25,30 Studies of swelling
behavior of the CXL-treated cornea17 reveal minimal
hydration of the anterior stroma compared with the rest
of the cornea. Wollensak et al31,32 described ultraviolet
A dose-dependent keratocyte apoptosis in a rabbit model,
with greater intensities destroying keratocytes deeper in the
stroma. They used 3-mW/cm2 ultraviolet A irradiance in
the human protocol, which affected keratocytes up to
a depth of 300 mm. Their studies of wound healing33 after
CXL demonstrated replacement of damaged stromal
keratocytes after 4 to 6 weeks by migrating peripheral
keratocytes. Esquenazi et al22 demonstrated the cellular
events and the ultrastructural modications of CXL in
rabbit corneas on immunostaining. Early keratocyte
necrosis and apoptosis were noted in the anterior stroma
after CXL, with early myobroblast transformation
beneath the treated area followed by gradual repopulation
of the anterior stroma.

2416

Botts et al24 used immunouorescence microscopy

to assess the effect of CXL on stromal collagen
in edematous corneas in humans. They evaluated
CXL-treated edematous corneas obtained from 7 patients
who underwent keratoplasty more than 6 to 90 days after the
procedure. The study included 1 patient at 7 days, 2 patients
at 10 days, 1 patient at 30 days, and 3 patients at 90 days
after CXL. Cross-linked corneas had a pronounced anterior
zone of organization and compaction of collagen bers on
immunouorescence microscopy. Nuclear staining with
DAPI demonstrated nuclear fragmentation in the anterior
stroma that lasted up to 3 months. The corneas with mild
disease showed more compaction than those with advanced
disease, but the effect was found to decrease with time. They
did not give any details of clinical features after CXL in
these patients.
Our study demonstrated stromal compaction after CXL
in 50% (12/24) of cases. The effect was seen in 58.6% of
cases at 1 month and in 41.3% cases at 3 months and was
evident histopathologically more at 1 month compared with
samples at 3 months. Stromal compaction also was
increased in moderate disease compared with the advanced
cases in both groups. The reduced effect in advanced cases
probably was the result of reduced riboavin diffusion into
the thicker stroma. The compaction of stromal collagen,
combined with increased hydrostatic and osmotic resistance
to uid accumulation after CXL, demonstrated that the
technique is useful in reducing corneal edema.
Keratocyte loss known to occur after CXL probably was
replaced by the repopulation of anterior stroma as early as 1
month after CXL. Hence, a relatively uniform distribution of
keratocytes was seen in the study. Loss of nucleus or its
fragmentation on DAPI staining was not noted in any
sample in our study. This is in contrast to other studies in
which nuclear fragmentation seen in the anterior, treated
part of the stroma immediately after CXL was retained for
up to 3 months. The relatively advanced corneal edema in
most of our cases could be the reason for decreased effect on
nuclear fragmentation.
Our results suggest that CXL in PBK decreases corneal
edema in patients with moderate to severe disease. A limitation of the study is that patients with early manifestation of
PBK were not included. The effect is at its maximum at 1
month and starts waning thereafter, although it is still
present at 3 months. Combining the clinical and histopathologic results in our study, it is evident that CXL-induced
stromal compaction is more evident in moderate compared
with advanced stages of the disease. For maximum benet,

Arora et al

&

CXL in Pseudophakic Bullous Keratopathy

it should be offered as a temporary therapeutic measure
early in the course of disease in patients awaiting
keratoplasty.

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Figure 2. Immunouorescence microscopy photographs of corneal buttons

in group A (AeD) and group B (EeH) under '10 magnication: (A, C,
E, G) stained with mouse antitype 1 collagen antibody and Alexauor 488
tagged antimouse immunoglobulin G, and (B, D, F, H) nuclei stained with
40 ,6-diaminido-2-fenilindole dihydrochloride (Molecular Probes, Carlsbad,
CA). AS anterior stromal compaction; KN keratocyte nucleus.

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Footnotes and Financial Disclosures

Originally received: November 27, 2012.
Final revision: July 18, 2013.
Accepted: July 22, 2013.
Available online: August 15, 2013.

Presented as a poster at: American Academy of Ophthalmology Annual

Meeting, November 2012, Chicago, Illinois.
Manuscript no. 2012-1728.

1
Guru Nanak Eye Centre, Maulana Azad Medical College, New Delhi,
India.
2

Department of Pathology, G. B. Pant Hospital, Maulana Azad Medical

College, New Delhi, India.

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Financial Disclosure(s):
The author(s) have no proprietary or commercial interest in any materials
discussed in this article.
Correspondence:
Gaurav Goyal, MS, 63, Savarkar Apartments, IP extension, Patparganj,
New Delhi 110092, India. E-mail: dr.gaurav.mamc@gmail.com.