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Contact Lens and Anterior Eye xxx (2016) xxxxxx

Contents lists available at ScienceDirect

Contact Lens and Anterior Eye

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

The impact of lens care solutions on corneal epithelial changes during

daily silicone hydrogel contact lens wear as measured by in vivo
confocal microscopy
Xiaolin Zhanga , Christine Marchettia , Jessica Leea , Yan Suna , Sara Debanneb , Ying Jiangb ,
Jami Kernc , Mark Harroda , Beth Ann Benetza , Eric Pearlmana,d , Loretta Szczotka-Flynna,*
a
Department of Ophthalmology & Visual Sciences and University Hospitals Eye Institute, University Hospitals Case Medical Center, Cleveland, OH, United
States
b
Department of Epidemiology & Biostatistics, Case Western Reserve University, Cleveland, OH, United States
c
Alcon Research, Fort Worth, TX, United States
d
Department of Ophthalmology, University of California, Irvine, United States

A R T I C L E I N F O A B S T R A C T

Article history: Purpose: To assess corneal epithelial microstructure via confocal microscopy and determine if cellular
Received 3 March 2016 changes are associated with lens care solutions during daily wear of silicone hydrogel contact lenses.
Received in revised form 9 November 2016 Methods: Corneal in vivo confocal microscopy with the Nidek ConfoScan4 was performed at baseline and
Accepted 11 November 2016
after 5 months of lotralcon A daily contact lens wear. Enrolled participants were randomized to use
either a polyhexamethylene biguanide (PHMB) preserved multipurpose care solution (MPS) or a peroxide
Keywords: based solution system. Lens and storage case bioburden were assessed with aerobic culture methods.
Confocal microscopy
Univariate and multivariable analyses were done to evaluate the association between solution use, or
Silicone hydrogel contact lens
Hyper-reective corneal epithelial cells
solution-related clinical covariates, and morphologic differences (hyper-reectivity) in the supercial
epithelial cells and epithelial basal cell density.
Results: Data on 139 participants were available for analysis of supercial epithelial cells while data on 92
participants were available for epithelial basal cell density. Five months after randomization to the
solution groups, 33% of participants had visible hyper-reective cells. More participants using MPS had
1 hyper-reective cells compared to peroxide users at 5 months (44% vs. 22%; p = 0.006). Similarly at 5
months, more participants with solution-induced corneal staining (SICS) had 1 hyper-reective cells
compared to non-SICS participants (57% vs. 29%; p = 0.010). The adjusted odds ratios (ORs) for risk of
presenting with hyper-reective cells in MPS users or SICS participants was 2.7 (95% CI; 1.275.65) and
3.4 (95% CI; 1.298.97), respectively. Basal cell density decreased by over 350 cells/mm2 over time (about
6%) in participants who had substantial bioburden on their lenses or in their storage case.
Conclusion: The confocal microscope can detect epithelial cellular changes in vivo during contact lens
wear. Hyper-reective supercial epithelial cells are associated with a PHMB preserved solution and
decreases in basal epithelial cell density may be associated with bacterial bioburden.
2016 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved.

1. Introduction cornea have not been systematically addressed. We examined

There is a wealth of literature evaluating the effects of lens care
solutions and microbial bioburden on corneal structure and
function [17]. However, the effects of multiple clinical covariates
during contact lens wear including solution type, lens or solution-
induced corneal staining, and bioburden on cellular changes in the
* Corresponding author at: University Hospitals Eye Institute, 11100 Euclid Ave
Lakeside 4126C, Cleveland, Ohio 44106, United States.
E-mail address: loretta.szczotka@uhhospitals.org (L. Szczotka-Flynn).
these variables using in vivo confocal microscopy as a non-invasive
way to examine the corneal epithelium at the cellular level.
Confocal microscopy is clinically used to diagnose atypical and
rare corneal infections [810] and corneal endothelial density and
morphology [11,12]. In the research setting, it is additionally used
to evaluate corneal epithelial structure and reectivity [13], sub-
basal nerve plexus [14], stromal reectivity [15], and pre-clinical
stromal corneal inltrates [16,17]. In vivo confocal microscopy can
also examine corneal layers during contact lens wear, after
refractive surgery, and determine the effect of immune-altered
disease states [1820].

http://dx.doi.org/10.1016/j.clae.2016.11.006
1367-0484/ 2016 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: X. Zhang, et al., The impact of lens care solutions on corneal epithelial changes during daily silicone hydrogel
contact lens wear as measured by in vivo confocal microscopy, Contact Lens & Anterior Eye (2016), http://dx.doi.org/10.1016/j.clae.2016.11.006
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2 X. Zhang et al. / Contact Lens & Anterior Eye xxx (2016) xxxxxx
Some of the more recent applications of confocal microscopy
have been in the analysis of the corneal epithelium. For example,
mean diameter of supercial epithelial cells have been shown to
increase with age using confocal microscopy [21]. In clinical disease,
confocal microscopy has been used to assess corneal epithelial
changes after refractive surgery and collagen crosslinking [22], and
Gabbriellini et al. recently assessed epithelial integrity, density, and
sub-basal plexus morphology in Sjgren's syndrome patients using
confocal microscopy [23]. Of relevance to this study, confocal
microscopy was used by Situ et al. to study hyper-reective corneal
epithelial cells during and after contact lens use [24], and Harvey
et al. found hyper-reective supercial corneal epithelial cells in
participants exposed to a specic contact lens/solution combination,
and suggested a correlation between solution-induced corneal
staining (SICS) and hyper-reective cells [25].
Due to the physical proximity of contact lenses on the ocular
surface, the corneal epithelium, as opposed to the deeper stromal
or endothelial layers, is most likely to demonstrate morphological
changes with lens wear. Two indices have been proposed capable
of detecting cell stress at the epithelial corneal surface; supercial
epithelial cell hyper-reectivity and basal cell density [23,25]. We
utilize them here as a mechanism to detect corneal cellular
changes secondary to variables related to daily contact lens wear
including use of care solutions and microbial contamination of
lenses. Specic goals of the current study were to use confocal
microscopy to determine whether contact lenses in combination
with care solutions over ve months of wear impacted the corneal
epithelium, controlling for confounding effects of other stress
factors, such as microbial bioburden, when possible.
2. Materials and methods
This was an ancillary study to the Daily Wear Corneal Inltrative
Event (DWCIE) Study, which was a prospective, investigator masked,
cohort study with an embedded randomized clinical trial of solution
use. In the DWCIE Study, 218 participants were t to the Air Optix 1
Night & Day1 Aqua Lens (lotralcon A, Alcon, Duluth, GA) for up to
30 days of daily wear with monthly disposal, and followed for 1 year
at the University Hospitals Eye Institute. The DWCIE and all ancillary
studies were approved by the University Hospitals Case Medical
Center Institutional Review Board and followed all the Tenets of the
Declaration of Helsinki. All participants provided written informed
consent prior to participation.
At the baseline DWCIE study visit, enrolled participants were
block randomized to one of two solutions: a preserved multipur-
pose (MPS) solution (Renu Fresh, Bausch & Lomb, Rochester, NY) or
a 1-step hydrogen peroxide care system (Clear Care1; Alcon, Fort
Worth, TX) using rub and rinse regimens with companion
rewetting drops. No wash out period from previous lens wear
was required. At baseline, 50% of participants habitually used a
PHMB-preserved MPS care product, 21% used a polyquaternium-
preserved MPS product, and 10% reported using a peroxide based
care product. Enrolled participants returned for follow-up visits
after 2 weeks, and after 1, 2, 5, 8 and 12 months of daily wear;
however, in the ancillary study, confocal microscopy images were
taken only at the baseline and 5 month visit, therefore, data up to
the 5 month visit are used in this analysis. A modied version of the
Institute for Eye Research (IER) grading scale was used for corneal
staining to quantify staining density in each of 5 corneal zones, as
was done in our previous studies [26]. Solution-induced corneal
staining (SICS) was dened as present when micropunctate stain
encompassing more than 15% surface area was present in at least 4
zones.
Aerobic cultures were routinely performed on the ocular
surface (lid margins and conjunctival surface), worn contact
lenses and storage cases as previously published [27]. In this
Please cite this article in press as: X. Zhang, et al., The impact of lens care solutions on corneal epithelial changes during daily silicone hydrogel
contact lens wear as measured by in vivo confocal microscopy, Contact Lens & Anterior Eye (2016), http://dx.doi.org/10.1016/j.clae.2016.11.006
analysis, bacterial bioburden from lenses collected after 1 and 5
months of wear and storage cases collected after 2 and 5 months of
use were assessed as exploratory covariates potentially impacting
epithelial cellular changes. Bacterial bioburden was dened as
substantial if there was presence of high levels of commensal
ocular biota or organisms of low pathogenicity, and/or moderate
levels (10 colony forming units (CFU)) of pathogenic organisms.
The levels of coagulase negative staphylococci (CNS) considered to
be substantial varied by the device cultured. If the CFU count of
CNS cultured from the lenses was 10, it was considered
substantial. If the CFU count of CNS cultured from the case or
residual case uid was 60, it was considered substantial.
2.1. Confocal microscopy
The ConfoScan4 (Nidek Technologies, Srl, Padova, Italy) and 40X
probe with a gel immersion exam was used in this study. All
participants had a baseline scan at the initial visit and again at the 5
month visit, to allow a sufcient time period of use in the lens and
care products to detect cellular changes. Scans were done by a
single ophthalmic photographer in the University Hospitals Eye
Institute. Scans were done at the end of each study visit with a daily
disposable 9.0 mm +0.50 D One-Day Acuvue (Vistakon, Jackson-
ville, FL) lens worn during the probe immersion exam to lessen
epithelial trauma to the subject and provide better epithelial
images [28]. Following placement of a drop of the topical
anesthetic (proparacaine) and placement of a bandage contact
lens, a drop of Genteal1 gel (Alcon, Fort Worth, TX) was placed on
the central cornea. Topical anesthetic was used as we found that
participants remained more comfortable and were more coopera-
tive when imaging both eyes, which ultimately resulted in better
images, and topical proparacaine has not been shown to induce any
cellular changes on confocal microscopy [13]. The objective piece
of the confocal microscope was placed in contact with the gel.
Three complete scans of the entire central cornea from the
endothelium to the epithelium in 5 mm steps were obtained from
each eye. The images were electronically stored and sent to the
Cornea Image Analysis Reading Center (CIARC) for masked
evaluation. The CIARC identied the best image(s) for each
respective corneal layer, saved and labeled these images for
masked analysis.
Epithelial images were analyzed by masked, experienced
readers. The number of epithelial hyper-reective cells were
independently read and counted by authors XZ and CM, who were
masked to solution group and all other covariates. In the
adjudication process, a third masked reader, author JL, reviewed
the ndings of the two masked readers. If the number of epithelial
hyper-reective cells as read by the rst two readers differed by <5
cells per eye-visit, the nal number of cells was taken as the
average of the 2 readings. When differences between the counts
were >5, the adjudicator re-interpreted the ndings and entered
the nal count. If the images were unanalyzable they were
excluded from analysis. Fig. 1 displays a representative image of
supercial epithelial hyper-reective cells.
The CIARC imported the confocal images into Konan Analysis
software (Konan KSS300 S v2.62, Konan Medical, Inc.). The Konan
Center method was used to analyze epithelial basal cell density.
Two masked readers (adjudicated by third in cases of >5%
discrepancy) selected the largest area of contiguous cells in the
image where the centers could be accurately distinguished, and
marked the center of all contiguous, analyzable cells. One to three
unique images were analyzed per time point to maximize the total
area and number of cells analyzed per time point. The software
then calculated the mean epithelial basal cell density and standard
deviation (cells/mm2). Fig. 2 displays a representative image of
epithelial basal cells.
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Fig 1. Representative image of epithelial hyper-reective cells.
2.2. Denition of clinical covariates and statistical analysis
Multiple clinical covariates (Table 1) were analyzed to
determine if there was any effect on the supercial or basal
epithelium as viewed by confocal microscopy. These covariates
were drawn from any visit up to and including the 5 month visit
and considered positive if present on at least one of these visits.
Outcome measurements were drawn from one eye of each patient.
Fig. 2. Representative image of epithelial basal cells.
Please cite this article in press as: X. Zhang, et al., The impact of lens care solutions on corneal epithelial changes during daily silicone hydrogel
contact lens wear as measured by in vivo confocal microscopy, Contact Lens & Anterior Eye (2016), http://dx.doi.org/10.1016/j.clae.2016.11.006
3
For basal epithelial cell density, the change between the 5 month
and baseline values were calculated. For analysis of supercial
epithelial cell hyper-reectivity, only the 5 month measurements
were compared because the outcome counts were very low with
probable variation in the corneal point analyzed between the two
visits, and thus a change value would not be clinically meaningful.
We assessed if the number of hyper-reective cells or change in
basal epithelial cell density signicantly differed when stratied by
the covariates in Table 1. Univariate comparisons were done using
the t-test for normally distributed data; otherwise, the non-
parametric Wilcoxon test was used. Hyper-reective cell counts
were also stratied into a binary covariate (no hyper-reective
cells or 1 hyper-reective cells) and used in univariate or
multivariate logistic regression. The chi-square test and Fishers
exact test were also used for comparisons of categorical variables.
Signicant covariates (p < 0.05) and biologically plausible cova-
riates of age and sex were then entered into multivariable linear or
logistic regression models; solution use and SICS are known to be
highly correlated therefore both were not used in the same model
due to potential multicollinearity. P-values were adjusted for
multiple uses of the data. All analyses were done using SAS 9.3.
3. Results
Baseline demographics of the overall cohort and results of the
main analyses have been published [27]. In brief, the participants
represented standard contact lens wearers found in routine clinical
practices. Participants eyes were clinically normal with no signs of
anterior segment disease or abnormal slit lamp signs. The mean
age of participants was 34.7 years (range 16.5 66.3) and 50
participants (22.9%) were 25 years of age. The majority of
participants were female (70%), Caucasian (59%), and existing or
recent lens wearers (88%). Almost half (44%) of participants had
experienced previous adverse events with contact lens wear,
which were equally distributed between the lens care groups by
design via block randomization. Of the 218 participants in the
parent DWCIE Study, 215 had baseline and 165 had 5-month in vivo
confocal microscopy examinations. Fig. 3 outlines the ow of the
participants and their available images throughout this ancillary
study by lens care group. Data on 139 participants were available
for analysis of supercial epithelial cells, while data on 92
participants were available for epithelial basal cell density
analyses.
3.1. Supercial epithelial cell hyper-reectivity
For the analysis of supercial epithelial cell hyper-reectivity,
there were 135 and 139 participants with readable images
available at the baseline and 5 month visits, respectively. Fig. 3
lists numbers of available images by solution group. The mean
number of hyper-reective cells within the best scan (representing
a 460  345 mm inspected eld in the central cornea) for all
participants was 0.45 (range 08) and 0.56 (range 06) at the
baseline and 5 month visits, respectively. The majority of
participants had zero hyper-reective cells visible at baseline
(69%) or 5 months (67%). Univariate analyses showed statistically
signicant differences in number of hyper-reective cells when
stratied by solution type, presence of SICS staining, and lens
bioburden. Table 2 provides the baseline and 5 month data for
mean number of supercial hyper-reective cells counted in each
central scan for staining, bioburden and solution covariates. At the
5 month visit, MPS users and SICS stainers had a greater mean
number of hyper-reective cells compared to the peroxide users
and non-SICS stainers, respectively. There were no differences
noted between the randomized solution groups at baseline, prior
to randomization, however, SICS stainers at baseline had more
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Table 1
Clinical Covariates.

Corneal Staining
Solution Induced Corneal type Staining (SICS) vs. not
Overall Grade 2 Corneal Staining vs. not
Two or more visits (over 5 months) with increased corneal staining compared to baseline
Two or more visits (over 5 months) where less corneal staining was noted compared to baseline

Conjunctival Staining
Grade 1 Circumlimbal lissamine green staining vs. not
Grade 2 Circumlimbal lissamine green staining vs. not
Grade 1 General lissamine green staining vs. not
Grade 2 General lissamine green staining vs. not

Microbiology
Substantial bioburden on lids, conjunctivae, lenses or cases vs. not
Substantial CNS or gram positive bioburden on lids or conjunctivae vs. not
Normal bioburden on lids or conjunctivae at 5 months and substantial bioburden at baseline
Substantial bioburden on lids or conjunctivae at 5 months and normal bioburden at baseline

Solution Use
MPS vs. Peroxide

DWCIE Baseline/Randomization visit (n = 218)

Confocal microscopy examinations (n=215)

Preserved multipurpose (MPS) solution Hydrogen peroxide care system

(n=107) (n=108)

BASELINE: BASELINE
Readable superficial epithelial images (n=67) Readable superficial epithelial images (n=68)
Readable basal epithelial cell images (n =62 ) Readable basal epithelial cell images (n =54 )

5 month visit (n=82) 5 month visit (n=83)

Readable superficial epithelial images (n=71) Readable superficial epithelial images (n=68)
Readable basal epithelial cell images (n =63) Readable basal epithelial cell images (n = 64)

Number of paired basal cell image sets Number of paired basal cell image sets
available (n =47) available (n =45)

Fig. 3. Flow of participants and their available images by lens care group.

hyper-reective cells. Substantial lens bioburden was associated
with a lower mean hyper-reective cell count. Chi-square analyses
were then performed to determine if there were differences in the
number of participants with 1 hyper-reective cells within these
same covariates. Only solution type and SICS staining remained
signicantly associated with presence of hyper-reective cells.
More participants using MPS had 1 hyper-reective cells in either
eye compared to peroxide users at 5 months (44% vs. 22%;
p = 0.006). Similarly at 5 months, more SICS participants had 1
hyper-reective cells compared to non-SICS participants (57% vs.
29%; p = 0.010).
To ascertain whether solution use and presence of SICS
remained associated with increased hyper-reectivity after
controlling for potentially confounding biologically plausible
covariates, multivariate logistic regression was used. After
controlling for age and sex, the MPS and presence of SICS remained
signicantly associated with the presence of hyper-reective cells
(Table 3). Participants who used the MPS were 2.7-fold more likely
to have hyper-reective cells while presence of SICS was associated
with a 3.4-fold increased odds of having hyper-reective cells.
Recognizing that SICS is most prevalent in the corneal
periphery, and the confocal probe is placed centrally, we assessed
SICS in the central corneal zone at the 5 month visit for association
with hyper-reectivity at this visit using Fisher exact tests. Central
SICS stainers were dened as those that had >45% surface area of
micropunctate staining in the central corneal zone. Of the 6
participants that had evidence of central SICS and usable
supercial confocal images at the 5 month visit, 5 of them (83%)

Please cite this article in press as: X. Zhang, et al., The impact of lens care solutions on corneal epithelial changes during daily silicone hydrogel
contact lens wear as measured by in vivo confocal microscopy, Contact Lens & Anterior Eye (2016), http://dx.doi.org/10.1016/j.clae.2016.11.006
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Table 2
Comparisons of Supercial Hyper-reective Epithelial Cells at Baseline and 5 Months for Solution Group and Solution Induced Corneal Staining.

Covariate of Interesta Time Point Mean (standard deviation) with sample sizeb available for each comparison and timepoint P value

Randomized Solution Group MPS Peroxide

n = 67 at baseline n = 68 at baseline
n = 71 at 5 months n = 68 at 5 months
Baseline 0.40 (0.76) 0.51 (1.25) 0.780
5 months 0.70 (1.22) 0.32 (0.82) 0.007
Solution induced corneal staining YES NO
n = 25 at baseline n = 110 at baseline
n = 21 at 5 months n = 118 at 5 months
Baseline 0.72 (1.02) 0.40 (1.03) 0.010
5 months 1.10 (1.65) 0.41 (0.88) 0.009
Overall Grade 2 Staining YES NO
n = 43 at baseline n = 92 at baseline
n = 45 at 5 months n = 94 at 5 months
Baseline 0.59 (1.44) 0.40 (0.78) 0.782
5 months 0.60 (0.99) 0.47 (1.09) 0.128
Improved Corneal Staining Over Time YES NO
n = 8 at baseline n = 127 at baseline
n = 10 at 5 months n = 129 at 5 months
Baseline 0.25 (0.38) 0.47 (1.06) 0.968
5 months 0.35 (0.94) 0.53 (1.06) 0.377
Worsened Corneal Staining Over Time YES NO
n = 27 at baseline n = 108 at baseline
n = 33 at 5 months n = 106 at 5 months
Baseline 0.31 (0.71) 0.50 (1.10) 0.324
5 months 0.74 (1.40) 0.44 (0.92) 0.202
Signicant lens bioburden YES NO
n = 19 at baseline n = 116 at baseline
n = 21 at 5 months n = 118 at 5 months
Baseline 0.32 (0.73) 0.48 (1.08) 0.368
5 months 0.10 (0.26) 0.59 (1.12) 0.029
Signicant case bioburden YES NO
n = 57 at baseline n = 67 at baseline
n = 61 at 5 months n = 68 at 5 months
Baseline 0.47 (1.22) 0.45 (0.90) 0.640
5 months 0.52 (1.07) 0.50 (1.01) 0.827
a
covariates refer to variables dened in Table 1 drawn from data across the 5 months of the study period.
b
one eye per participant was analyzed thus sample sizes refers to participants or eyes.

Table 3
Multivariate Logistic Regression Models with Hyper-reective Cells (1 cell) at 5 months as Outcome.

A: Model Exploring Solution Use as Covariate, signicant covariate in bold

Covariate Odds Ratio 95% Wald Condence Limits P value
Randomized Solution Group 2.68 1.27 5.65 0.010
(multipurpose vs. peroxide)
Age 0.81 0.34 1.96 0.645
(25 vs. >25)
Sex 0.63 0.27 1.44 0.271
(male vs. female)

B: Model Exploring Solution Induced Corneal Staining (SICS) as Covariate; signicant covariate in bold
Covariate Odds Ratio 95% Wald Condence Limits P value
SICS 3.40 1.29 8.97 0.013
(yes vs. no)
Age 0.74 0.31 1.8 0.511
(25 vs. >25)
Sex 0.60 0.26 1.4 0.229
(male vs. female)

had 1 hyper-reective cells compared to 37/141 (26%) of non-
central SICS participants, and this difference was signicant
(p = 0.008).
3.2. Basal epithelial cell density
As shown in Fig. 3, there were 92 participants with paired
images from both the baseline and 5 month time-points; only
these 92 participants that had usable paired images were used in
the basal cell analyses. Univariate associations between covariates
of interest in Table 1 and basal epithelial cell density are shown in
Table 4. Improved corneal staining was associated with a change
(increase) in epithelial basal cell density from baseline to 5 months.
This table can be interpreted as follows; there was an increase in
epithelial basal cell density from baseline to 5 months (by a mean
of 585 cells/mm2) in the group that showed improved corneal
staining over the rst 5 months of the study compared to those that
did not. Participants with substantial storage case bioburden
through the 5 month visit had a signicant decrease in mean basal
cell density over time. The multivariate linear regression model

Please cite this article in press as: X. Zhang, et al., The impact of lens care solutions on corneal epithelial changes during daily silicone hydrogel
contact lens wear as measured by in vivo confocal microscopy, Contact Lens & Anterior Eye (2016), http://dx.doi.org/10.1016/j.clae.2016.11.006
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Table 4
Change in Basal Epithelial Cell Density from Baseline to the 5 Month Visit by Various Covariates of Interest.

Covariate of Interest Mean (standard deviation)* P value

Randomized Solution Group MPS Peroxide

n = 47 n = 45
58.45 (623.26) 43.41 (740.10) 0.507
Solution induced corneal staining YES NO
n = 14 n = 78
43.03 (615.88) 2.46 (695.60) 0.892
Overall Grade 2 Staining YES NO
n = 32 n = 60
58.77 (638.39) 18.11 (706.52) 0.443
Improved Corneal Staining Over Time YES NO
n=4 n = 88
584.88 (89.58) 17.56 (684.77) 0.030
Worsened Corneal Staining Over Time YES NO
n = 22 n = 70
15.5 (543.82) 6.47 (722.28) 0.982
Signicant lens bioburden YES NO
n = 15 n = 77
17.48 (1067.12) 13.72 (587.74) 0.305
Signicant case bioburden YES NO
n = 36 n = 49
261.16 (628.76) 173.21 (684.74) 0.005

Change value derived by subtracting baseline value from the 5 month value, positive values indicate increased cell density over time.

(Table 5) including age and sex found that only abnormal
microbiology within storage cases remained independently
associated with basal cell density changes from baseline to 5
months. Specically, the basal cell density decreased by 366 cells/
mm2 (about 6%) in those participants with storage case bioburden.
Analysis of solution and other staining variables did not show
signicant effects or trends on change in basal epithelial cell
density (p > 0.100) when controlling for storage case bioburden.
Although lens bioburden was not signicant in univariate analysis,
multivariate analysis showed similar trends to storage case
bioburden. That is, basal cell density decreased by 416 cells/
mm2 in those participants with substantial lens bioburden
(p=0.017) and in this model, improved corneal staining was
associated with increased epithelial basal cell density (p = 0.025)
4. Discussion
We used confocal microscopy to assess hyper-reectivity of the
supercial corneal epithelial cells, which has been reported in
several studies to be associated with various contact lens and
solution combinations [13,29]. We found that the presence of
hyper-reective supercial epithelial cells was associated with a
PHMB-preserved MPS as well as SICS. We calculated that MPS
users were 2.7 fold more likely to have hyper-reective cells
compared to the peroxide group whereas presence of SICS was
associated with a 3.4 fold increased risk of having hyper-reective
cells. These covariates could not be assessed independently and we
believe the mechanism of preservative induced corneal staining
and epithelial cell hyper-reectivity are probably linked.
Hyper-reective corneal epithelial cells have not been exten-
sively reported or studied in the literature and to our knowledge
this is the rst published study on the link between hyper-
reective cells and solution use although others have previously
found the same association [13,29]. Scanning electron microscopy
and confocal microscopy have identied different shades of
supercial cells. Dark cells possess fewer surface features, and
are assumed to have lost microvilli [3032]. Lighter cells may be
the most recently recruited to the supercial epithelial cell layer,
because they have surface features consistent with microvilli as it
has been proposed that microdesmosomes could explain brighter
cell membranes of epithelial cells [33,34]. Therefore, darker cells
are presumed to represent mature cells that are in process of being
desquamated [30,32]. In that regard, Wilson et al. suggested that
differences in cytoplasm reectivity could represent stages of
progression towards cell death, with the less reective or darker
cells about to desquamate [35].
Hyper-reective cells are different than the lightly shaded
epithelial cells previously described using confocal microscopy. In

Table 5
Linear Regression Models with Basal Epithelial Cell Density Change from Baseline to 5 months as Outcome, signicant covariates in bold.

Covariate Estimate 95% CI P value

Improved corneal staining vs. not 564.50 188.3 to 1317.2 0.142
Substantial bacterial bioburden on cases vs. not 366.70 649.5 to 83.9 0.011
Age 156.00 481.5 to 169.5 0.348
(25 vs. >25)
Sex 162.60 439.2 to 113.9 0.249
(male vs. female)

Covariate Estimate 95% CI P value

Improved corneal staining vs. not 708.77 87.09 to 1330.45 0.025
Substantial bacterial bioburden on lenses vs. not 416.17 -758.21 to 74.13 0.017
Age 163.51 461.66 to 134.64 0.282
(25 vs. >25)
Sex 135.12 392.51 to 122.27 0.304
(male vs. female)

Please cite this article in press as: X. Zhang, et al., The impact of lens care solutions on corneal epithelial changes during daily silicone hydrogel
contact lens wear as measured by in vivo confocal microscopy, Contact Lens & Anterior Eye (2016), http://dx.doi.org/10.1016/j.clae.2016.11.006
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known circumstances of ocular surface disease, hyper-reective
cells (in various locations within corneal tissue) have been linked
to pro-inammatory states such as viral keratitis [36,37], pterygi-
um [38], vernal keratoconjunctivitis [39], Salzmanns nodular
degeneration [40], and ocular changes in systemic inammatory
states such as Crohns disease[41] and Stevens Johnsons Syndrome
[42]. Hamrah et al. proposed that an increase in hyper-reective
epithelial cells in patients with herpes simplex viral keratitis is due
to supercial epithelial cell desquamation [37]. Outside of active
ocular surface disease, hyper-reectivity has also been shown to be
associated with dry eye and normal cellular turnover [43,44].
Additionally, hyper-reective supercial epithelial cells have been
associated with use of certain preserved anti-glaucoma eye drops
and benzalkonium chloride [4547]. Therefore, although there is
little data on mechanisms causing hyper-reectivity of supercial
epithelial cells, in general, hyper-reectivity has been postulated to
correlate with inammation or toxicity. When present on the
supercial cornea in greater numbers in patients that use
preserved anti-glaucoma eye drops or benzalkonium chloride,
its presumed the changes observed are a form of preservative
toxicity [4547]. Additionally, hyper-reective stromal keratocytes
as imaged on confocal microscopy are assumed to correspond to a
state of metabolic activation induced by pro-inammatory
cytokines [48,49]. Thus, although the signicance of hyper-
reective cells is not known, in other layers of the cornea, they
are presumed to correspond to a pro-inammatory state as well.
In contact lens wearers, Harvey et al. found hyper-reective
supercial corneal epithelial cells in participants exposed to a
specic contact lens/solution combination, and suggested a
correlation between SICS and hyper-reective cells [25]. Schneider,
expanded upon these ndings and found that normal non-contact
lens wearers had a few random supercial hyper-reective cells
which did not change over 9 months [13]. She found increased
numbers of hyper-reective cells associated with higher levels of
SICS in a PHMB-based solution compared to a polyquaternium-
based and hydrogen peroxide-based solution during short term
use (2 weeks-1 month) [13]. SICS has been observed with certain
combinations of contact lens solutions and silicone hydrogel lenses
[4,50], and has been hypothesized by some to be a result of a toxic
reaction [4,51]. SICS is typically represented by ne micro-
punctate uorescein staining of the supercial epithelium usually
in the corneal periphery, is typically asymptomatic, and is most
evident during the rst 24 h of lens wear. It is well established
that certain lens types in combination with the same polyhexa-
methylene biguanide-based solution we used in our study, is
associated with signicantly more SICS than other non-PHMB
preserved solutions due to uptake and release of preservatives into
the lens matrix [4,52], and hydrogen peroxide-based solutions are
associated with the least amount of SICS [32,51].
In our study, based on the fact that the percentage of
participants that have hyper-reective cells is greater in the
PHMB-based MPS and SICS participants, and much greater in
participants with central SICS, our data is consistent with the
hypothesis that the hyper-reective cells are likely representative
of the stained cells induced by the solution. Our ndings reect
that hyper-reective cells co-exist in participants with SICS. We
conclude that hyper-reective cells occurred more commonly in
our MPS users, especially in MPS users who exhibited SICS. The
hyper-reective cells may reect the same cells that will stain with
sodium uorescein.
Since sodium uorescein and topical anesthetic were used prior
to confocal microscopy in this study, it has been suggested that
these topical agents could be responsible for the hyper-reectivity.
In that regard, Mocan et al. suggested that sodium uorescein
damaged epithelial tight junctions which resulted in increased
hyper-reective cells from epithelial sodium uorescein diffusion
Please cite this article in press as: X. Zhang, et al., The impact of lens care solutions on corneal epithelial changes during daily silicone hydrogel
contact lens wear as measured by in vivo confocal microscopy, Contact Lens & Anterior Eye (2016), http://dx.doi.org/10.1016/j.clae.2016.11.006
7
[53]. However, Sindt et al., using laser-scanning confocal micros-
copy (which uses a wavelength of 670 nm), found that uorescein
staining did not interfere with confocal imaging of corneal
epithelium, sub-basal nerves, or dendritic immune cells [54].
Schneider, using white-light slit scanning confocal microscopy as
we did, reported no difference in cellular appearance between eyes
that had sodium uorescein from those without, implying that
sodium uorescein and its residue are not associated with hyper-
reective cells. In fact, even after provocatively inducing SICS, the
use of sodium uorescein prior to confocal microscopy did not
affect appearance of hyper-reective cells [13]. While topical
anesthetics have been associated with inhibition of corneal
epithelial migration and disruption of supercial corneal epithelial
microvilli [55,56], Schneider showed that topical anesthetic was
not linked to the appearance of hyper-reective cells because even
when confocal microscopy was done without anesthetic, hyper-
reective cells were observed [13].
Basal epithelial cell density was also evaluated with confocal
microscopy. These deep epithelial cells undergo mitosis with cell
differentiation as they move anteriorly. We found a mean epithelial
basal cell density across participants of 6147  171 cells/mm2. This
nding is consistent with previous reports; Schneider had
reviewed others work and reported basal epithelial cell density
using confocal microscopy ranged from 3601  408 cells/mm2 to
6188  427 cells/mm2 [13]. Participants with substantial bacterial
burden on their lenses or within their storage cases were
independently associated with decreased basal epithelial cell
density. Only one of our staining variables was associated with
changes in basal cell density. Participants with decreased corneal
staining over time were associated with an increase in their basal
epithelial cell density. In contrast, worsening of corneal staining
over time, SICS or overall staining were not associated with
decreases in basal cell density. Therefore, further research is
required in this regard to understand the relationship between
corneal staining and impact on basal epithelial cell density.
Corneal epithelial basal cell density has been identied as an
indicator of cell stress in other studies. Many of these studies
collectively suggest that inammatory conditions (atopic and
vernal keratoconjunctivitis, dry eyes, and overnight orthokeratol-
ogy) can result in decreased epithelial basal cell density [39,57
59]. In eyes with atopic keratoconjunctivitis and dryness, corneal
staining was found to be correlated with decreased basal epithelial
cell density [58]. Additionally, Zhang et al. discovered a signicant
decrease in basal epithelial cell density in mild, moderate, and
severe dry eye participants when compared with controls [58]. The
authors suggested that increased tear osmolarity elicits pro-
inammatory cytokines, which decrease basal cell density even
before disrupting epithelial barrier function. Hamrah et al. noted
that basal epithelial cell density was lower in herpes simplex viral
keratitis patients when compared with controls [37], and others
described a signicant decrease in basal epithelial cell density in
clear corneas adjacent to pterygium when compared with controls
[60]. Therefore, ocular surface inammation appears to be
associated with decreased epithelial basal cell density, and our
lens bioburden ndings support this theory.
One limitation of the study includes no washout period prior to
study entry. However, this should not inuence the hyper-
reective cell analyses because only the 5 month visit data were
used, thus not inuenced by habitual lens or solution type. The
baseline basal cell density may have been affected by habitual lens
use, and changes seen by 5 months could be inuenced by retting
all participants to a new lens type or solution system. However, we
controlled for factors associated with lens type or solution use at
baseline (staining and bacterial bioburden) in the linear regression
models (Table 5), therefore, outcomes of this analyses were not
confounded.
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8 X. Zhang et al. / Contact Lens & Anterior Eye xxx (2016) xxxxxx
5. Conclusions
Confocal microscopy can identify multiple cellular changes in
the cornea during daily contact lens wear. We have identied
potential cellular stresses on the ocular surface by recognition of
hyper-reective supercial epithelial cells and decreases in basal
epithelial cell density. We propose that SICS staining secondary to a
PHMB-preserved MPS results in hyper-reective supercial
epithelial cells noted during confocal microscopy. Furthermore,
lens microbial bioburden is associated with decreases in basal
epithelial cell density; one explanation may be that this represents
decreased proliferation or increased apoptosis of corneal basal
epithelial cells. Although these ndings are exploratory in nature
and deserve further study, the confocal microscope is a powerful
instrument that can detect cellular changes in vivo during contact
lens wear.
Source of funding
This work was supported by Alcon Research Ltd. with indirect
support for laboratories and coordination from the Ohio Lions Eye
Research Foundation and Research to Prevent Blindness. The
clinicalTrials.gov identier is NCT00937105.
Conicts of interest
Dr. LSF has received research support from Alcon Laboratories
and Johnson & Johnson Vision Care, Inc. in the last 12 months. J.
Kern is employed by Alcon Research.
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Please cite this article in press as: X. Zhang, et al., The impact of lens care solutions on corneal epithelial changes during daily silicone hydrogel
contact lens wear as measured by in vivo confocal microscopy, Contact Lens & Anterior Eye (2016), http://dx.doi.org/10.1016/j.clae.2016.11.006