sodium hyaluronate decreases ocular surface toxicity...

Glaucoma

Sodium Hyaluronate Decreases Ocular Surface ToxicityInduced by Benzalkonium Chloride–PreservedLatanoprost: An In Vivo Study

Fenfen Yu, Xing Liu, Yimin Zhong, Xinxing Guo, Mei Li, Zhen Mao, Hui Xiao, and Shasha Yang

State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China

Correspondence: Xing Liu, State KeyLaboratory of Ophthalmology,Zhongshan Ophthalmic Center, SunYat-sen University, Guangzhou510060, China;[email protected].

Submitted: October 21, 2012Accepted: April 18, 2013

Citation: Yu F, Liu X, Zhong Y, et al.Sodium hyaluronate decreases ocularsurface toxicity induced by benzalko-nium chloride–preserved latanoprost:an in vivo study. Invest Ophthalmol

Vis Sci. 2013;54:3385–3393. DOI:10.1167/iovs.12-11181

PURPOSE. To investigate the effects of sodium hyaluronate (SH) on ocular surface toxicityinduced by benzalkonium chloride (BAC)-preserved latanoprost.

METHODS. Twenty-one white rabbits (42 eyes) were randomly divided into three groups. Thecontrol group was untreated. The two experimental groups were treated with 0.02% BAC-containing latanoprost once a day combined with unpreserved 0.3% SH or PBS three timesdaily for 60 days. Schirmer test, fluorescein and rose bengal staining, and conjunctivaimpression cytology were performed on days 0, 31, and 61. Apoptosis of conjunctivalepithelium was detected by TUNEL assay on day 61. Conjunctival inflammation was evaluatedwith light microscopy. Cornea and conjunctiva ultrastructure were observed by electronmicroscopy.

RESULTS. Compared with the control group, the PBS-treated latanoprost group showedincreases in fluorescein and rose bengal scores, decreases in Schirmer scores, and goblet celldensity (GCD) on days 31 and 61. Increases in inflammatory and apoptotic cells inconjunctiva, and ultrastructural disorders of the cornea and conjunctiva were also observedon day 61. Compared with the PBS-treated latanoprost group, the SH-treated latanoprostgroup showed decreases in fluorescein and rose bengal scores, and increases in Schirmerscores and GCD on days 31 and 61. Decreases in inflammatory and apoptotic cells inconjunctiva and amelioration of ultrastructural disorders were also observed.

CONCLUSIONS. Topical application of SH significantly decreased the ocular surface toxicityinduced by BAC-preserved latanoprost. As a vehicle or neutralizing material, SH could beproposed to reduce ocular toxicity and protect the ocular surface in long-term BAK-preservedantiglaucoma medication treatment.

Keywords: sodium hyaluronate, ocular surface toxicity, benzalkonium chloride, latanoprost

Topical antiglaucoma medications, such as the prostaglandinanalogue latanoprost, are widely and preferably used to

control IOP in glaucoma management. Long-term use of thesedrugs is often associated with ocular surface toxicity, such asallergic reactions, dry eye symptoms, subconjunctival fibrosis,and increased rate of surgery failure. In the United States, a cross-sectional study evaluated that 59% of glaucoma patients reporteddry eye symptoms in at least one eye.1 Several lines of evidencedemonstrate that these medications are associated with reduc-tion of tear film break-up time and basal tear secretion1,2;decrease of goblet cell density3,4; development of subconjunc-tival fibrosis most likely related to an increase in inflammatorycells5; overexpression of HLA-DR, intercellular adhesion mole-cule 1 (ICAM-1), or IL-6, IL-8, or IL-10 in the epithelium6; andincreased apoptosis rates in the conjunctival epithelial cells.7

Multiple studies have indicated that the toxicity ofantiglaucoma eye drops is largely due to their preservative,benzalkonium chloride (BAC).8–10 BAC is one of the mostcommonly used preservatives because of its higher antimicro-bial efficiency, stability, and low cost. Usually its concentrationsrange from 0.004% to 0.02% in antiglaucoma eye drops.11 Toreduce the ocular surface toxicity and enhance compliance ofglaucoma patients, several approaches, including fixed combi-

nations (i.e., bimatoprost 0.03%/timolol 0.5%, travoprost0.004%/timolol 0.5%), unpreserved drops (mainly beta-block-ers), and application of less toxic preservatives, have beenmade in the recent past.5,12,13 However, higher cost, difficultyin handling, and requirement of longer-term observation oftheir antimicrobial effects have been pointed out.11 Anotherattempt is to provide some vehicle or neutralizing material thatwould limit toxic effects.

Sodium hyaluronate (SH) is a linear polymer built fromrepeating disaccharide units containing N-acetylglucosamine andglucuronic acid. The polymer is stabilized by hydrogen bondsparallel with the chain axis and behaves in solution as anexpanded random coil. The coil can be regarded as a highlyhydrated sphere containing approximately 1000-fold more waterthan polymer.14 Native hyaluronate is an important structuralelement of the extracellular matrix and widely exists in vertebratetissues, notably in the vitreous body of the eye ball. A minority ofstudies showed recently that SH significantly reduces BAC-induced cytotoxic effects in vitro,15 but further in vivo studiesare needed when extrapolating these findings to clinical settings.The purpose of this study was to investigate in vivo the ability ofSH to reduce the ocular surface toxicity induced by a commonlyused topical antiglaucomatous medication.

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MATERIALS AND METHODS

Animals and Treatments

All procedures were performed in accordance with the ARVOStatement for the Use of Animals in Ophthalmic and VisionResearch and were approved by the animal ethics committeeof Zhongshan Ophthalmic Center, Sun Yat-sen University(approval ID: 2010-028). Twenty-one female white NewZealand rabbits (2.5–3.0 months old, 2.0–2.5 kg, purchasedfrom Guangdong Medical Laboratory Animal Center, Guang-dong, China) were used. The rabbits were placed in cages (onerabbit per cage) and kept in standard conditions (roomtemperature 238C 6 28C, relative humidity 60% 6 10%, 12-hour/12-hour light/dark cycle) and fed on the standardlaboratory diet with free access to water throughout the study.Before all experiments, the ocular surface integrity wasexamined by slit-lamp microscope (Topcon, Tokyo, Japan).

In this study, we used one type of commercially availableunpreserved 0.3% SH eye drops (Hialid; Santen, Osaka, Japan)and one type of commercially available 0.005% latanoprost eyedrops (Xalatan; Pfizer, New York, NY) containing the highestconcentration of BAC (0.02%).

Twenty-one rabbits (42 eyes) were randomly divided intothree groups. The control group was untreated for baselinecomparison. The PBS-treated latanoprost group was adminis-tered with latanoprost eye drops once daily (10 PM) and 50 lLPBS (pH 7.4) three times daily (8 AM, 2 PM, 8 PM). The SH-treated latanoprost group was administered with latanoprosteye drops once daily (10 PM) and 50 lL 0.3% SH three timesdaily (8 AM, 2 PM, 8 PM).

In all groups, fluorescein staining, rose bengal staining,Schirmer test, and conjunctiva impression cytology (CIC) wereperformed sequentially following the methods describedbelow on days 0, 31, and 61. At day 61, all rabbits were killedwith an overdose of general anesthetics. The conjunctivalapoptosis was detected by in situ terminal deoxynucleotidyltransferase dUTP nick end labeling (TUNEL) assay. Conjuncti-val inflammation was evaluated with light microscopy.Histomorphological changes of cornea and conjunctiva wereobserved by light microscopy or scanning electron microscopy(SEM) or transmission electron microscopy (TEM).

Fluorescein and Rose Bengal Staining

One microliter of 1% fluorescein sodium was instilled in theconjunctival sac and then rinsed with PBS 1 minute later. Theeyes were examined and photographed under a slit-lampmicroscope with a cobalt blue filter and a digital camera(Topcon, Tokyo, Japan). Five corneal areas were considered,the area of positive corneal staining was scored from 0 (absent)to 3 (diffuse loss of epithelium).16

Thirty minutes later, the disappearance of staining wasobserved under a slit-lamp microscope. One microliter of 1%rose bengal was instilled into the conjunctival sac. Fifteenseconds later, the scores were graded according to the vanBijsterveld grading system under a slit-lamp microscope(Topcon).17

Measurement of Aqueous Tear Production

After intramuscular injection of a mixture of 50 mg ketamineand 25 mg chlorpromazine and topical application ofproparacaine (Alcaine; Alcon, Fort Worth, TX), tear productionwas measured by the Schirmer test using Whatman 41 filterpaper strip (Tianjin Jingming New Technology DevelopmentCo., Ltd., Tianjin, China) at a similar time point (9 AM). Thestrip was placed in the outer third of the lower eyelid, and the

wetted length of the strip was read after 5 minutes. Each eyewas tested 3 times, and the average length was considered asthe final length.

Conjunctival Impression Cytology

Following an intramuscular injection of a mixture of 50 mgketamine and 25 mg chlorpromazine and topical application ofproparacaine (Alcaine), two 3.5 3 3.5-mm round nitrocellulosefilter papers (Pall Corporation, New York, NY) were appliedseparately on the nasal and temporal bulbar conjunctiva andpressed for 10 seconds with constant pressure. The specimenswere fixed with 95% ethanol and stained with periodic acidSchiff (PAS) and hematoxylin reagents. The number of gobletcells was counted under a light microscope (Olympus, Tokyo,Japan) in a masked fashion. Three nonadjacent high-powerfields (HPFs, 3400) of each specimen were selected randomlyfor counting, and an average was calculated. The morphologyof the conjunctival epithelium was graded according to theNelson method.18

TUNEL Assay

End-stage apoptosis was detected by in situ TUNEL labeling onparaffin sections of the superior bulbar conjunctiva. Formalin-fixed and paraffin-embedded 5-lm-thick tissue sections weredewaxed and rehydrated in a graded ethanol series (100%,95%, 85%, 70%), rinsed with PBS, permeabilized by 0.1% TritonX-100 and 0.1% sodium citrate, incubated with terminaldeoxynucleotidyl transferase solution and converter-peroxi-dase according to the manufacturer’s instructions (in situ celldeath detection kit-POD; Hoffmann-La Roche Co. Ltd., Basel,Switzerland), immersed in 3,30-diaminobenzidine (DAB), andcounterstained with hematoxylin. The samples were observedand photographed under a light microscope. TUNEL-positiveapoptotic cells in the conjunctival epithelia were distinguishedby brown immunoreactivity.

Light Microscopy

The superior bulbar conjunctiva was fixed in 10% formalin.After dehydration in a graded ethanol series, the specimenswere embedded in paraffin and cross-sectioned. The histolog-ical changes were observed by hematoxylin-eosin staining. Thenumber of inflammatory cells in the conjunctival epitheliumand stroma were counted in a masked manner. Threenonadjacent HPFs (3400) of each specimen were selectedrandomly for counting, and an average was calculated.

Scanning Electron Microscopy

Corneas were fixed with 2.5% glutaraldehyde, postfixed withosmium tetroxide, dehydrated in a graded ethanol series, driedwith hexamethyldisilazane, sputter-coated with gold, andexamined under SEM (Quanta 200; Philips, Amsterdam, TheNetherlands).

Transmission Electron Microscopy

Cornea and conjunctiva were fixed with 2.5% glutaraldehyde,postfixed with osmium tetroxide, embedded in resin, cut insections with 60-nm thickness, then examined under TEM(Tecnai G2; Philips).

Statistical Analysis

Statistical analyses were performed using the SPSS softwareversion 13.0 (SPSS Inc., Chicago, IL). Repeated-measures ANOVA

Effects of SH on Ocular Surface Toxicity IOVS j May 2013 j Vol. 54 j No. 5 j 3386


(Bonferroni posttest) was applied to make comparisons ofaqueous tear production and conjunctival goblet cell densityamong the groups over the study period. One-way ANOVA(Bonferroni posttest) was applied to make comparisons ofconjunctival inflammation among the groups on day 61.Kruskal-Wallis test (Bonferroni posttest) was applied to makecomparisons of fluorescein and rose bengal staining scores. P

values less than 0.05 were considered statistically significant.

RESULTS

Fluorescein and Rose Bengal Staining

There were significant differences in fluorescein (Kruskal-Wallis test, H ¼ 31.078, P < 0.001) and rose bengal (Kruskal-Wallis test, H ¼ 28.470, P < 0.001) staining scores among thegroups on day 61. Compared with the control group,fluorescein (Bonferroni, P < 0.001, Fig. 1A) and rose bengal(Bonferroni, P < 0.001, Fig. 1B) staining scores increased in thePBS-treated latanoprost group on day 61. There was nosignificant difference of the scores between the SH-treatedlatanoprost group and the control group. In the SH-treatedgroup, fluorescein (Bonferroni, P < 0.001, Fig. 1A) and rosebengal (Bonferroni, P < 0.001, Fig. 1B) staining scores weresignificantly lower than the PBS-treated group.

Aqueous Tear Production

There were significant differences in aqueous tear production(repeated-measures ANOVA, F ¼ 5.72, P < 0.01) among thegroups over the study period. Compared with the controlgroup, aqueous tear production (Bonferroni, both P < 0.001)decreased in the PBS-treated latanoprost group on days 31 and61. There was no significant difference of aqueous tearproduction between the SH-treated latanoprost group andthe control group. In the SH-treated group, aqueous tearproduction (Bonferroni, both P < 0.01) was more significantlyincreased than in the PBS-treated group on days 31 and 61 (Fig.2A).

Changes of Goblet Cell Density and ConjunctivalEpithelium Morphology

There were significant differences in goblet cell density (GCD)(repeated-measures ANOVA, F ¼ 17.38, P < 0.001) among the

groups over the study period. Compared with the controlgroup, GCD decreased in both PBS-treated (Bonferroni, both P

< 0.001) and SH-treated (Bonferroni, both P < 0.05)latanoprost groups on days 31 and 61. In the SH-treated group,GCD (Bonferroni, P < 0.01, P < 0.001, respectively) was moresignificantly increased than in the PBS-treated group on days 31and 61 (Fig. 2B). Grading scores increased from 0 to 2 on day61 in the PBS-treated latanoprost group. Squamous metaplasiadeveloped with time shown as the flattening of conjunctivalepithelial cells and increase of nucleocytoplasmic ratio.However, grading scores were only 0 to approximately 1 inthe SH-treated latanoprost group (Fig. 3).

Inflammatory Cell Infiltration in Conjunctiva

There was a significant difference in the number of inflamma-tory cells (one-way ANOVA, F ¼ 74.62, P < 0.001) among thegroups on day 61. Compared with the control group, thenumber of inflammatory cells in the conjunctival epitheliumand stroma increased in both the PBS-treated (Bonferroni, P <0.001) and SH-treated (Bonferroni, P < 0.05) latanoprostgroups on day 61. In the SH-treated group, conjunctivalinflammatory cell infiltration (Bonferroni, P < 0.001) wassignificantly less than in the PBS-treated group on day 61 (Fig.4).

Apoptosis in Conjunctival Epithelia

TUNEL staining revealed marked apoptosis in the conjunctivalepithelium in the PBS-treated latanoprost group. However,apoptosis was visibly relieved in the SH-treated latanoprostgroup (Fig. 5).

Ultrastructural Changes of Cornea and

Conjunctiva

Under the SEM, the cornea showed many bright cells coveredwith high-density microvilli in the control group. In the PBS-treated latanoprost group, the cornea showed a large numberof dark cells covered with seriously destroyed microvilli,diffused epithelia exfoliation and deformation, and cytoplasmwrinkling. However, there were many bright cells coveredwith high-density microvilli and minimal cytoplasm wrinklingin the SH-treated latanoprost group (Fig. 6).

FIGURE 1. Alterations of fluorescein and rose bengal staining scores. (A) Compared with the control group and SH-treated latanoprost group,fluorescein staining scores increased in the PBS-treated latanoprost group (both P < 0.001) on day 61. (B) Compared with the control group and SH-treated latanoprost group, rose bengal staining scores increased in the PBS-treated latanoprost group (both P < 0.001) on day 61.



Under TEM, sharp and high-density microvilli could be seenon the outer surfaces of corneal epithelial cells in the controleyes and the SH-treated eyes. Intracellular structures such asmitochondria, Golgi apparatus, and rough endoplasmic retic-ulum were also clearly presented. In corneal epithelial cells ofthe PBS-treated latanoprost group, flat and low-densitymicrovilli, granular clumps in the nuclear chromatin, wideningof the perinuclear cisterna (PN), and swelling of intracellularstructures were observed (Fig. 7).

Similar changes were observed in conjunctival epithelialcells under TEM. Abundant microvilli on the epithelial cells andsecretory granules in the goblet cells could be seen in thecontrol group. In the PBS-treated latanoprost group, decreasedmicrovilli, nuclear chromatin condensation of the epithelialcells, and reduction of secretory granules in the goblet cellswere observed. These damages were greatly mitigated in theSH-treated latanoprost group (Fig. 8).

DISCUSSION

Hyaluronate is not only an important component of extracel-lular matrix but also interacts with cells during several cellularpathways, such as embryonic morphogenesis, inflammation,

and wound repair, as well as chemoresistance. It regulates cellmovement and transport of extracelluar components.19 Itcould inhibit nitric oxide–induced apoptosis and dedifferenti-ation of articular chondrocytes,20 and upgrade VEGF expres-sion in rabbit Achilles tendon.21 At present, SH is widely usedas a kind of substitute for tears. Clinical research has shownthat SH could improve grading scores of conjunctivalimpression cytology for treating severe dry eye in Sjogren’ssyndrome patients.22 In this study, we demonstrated that SHcould decrease ocular surface toxicity induced by benzalko-nium chloride–preserved latanoprost. To our best knowledge,this has been the first in vivo study to demonstrate the functionof SH to decrease the toxicity induced by BAC-preservedantiglaucoma drops.

Although quite a few studies suggested that the activecomponent latanoprost did not induce overexpression ofvarious inflammatory cytokines,23,24 and was even responsiblefor protective effects against BAC toxicity by its antioxidativeproperties, evidence showed that BAC-preserved latanoprostinduced toxicity on conjunctival epithelial cells in vitro.25 Wedemonstrated significant time-dependent ocular surface toxic-ities in 0.02% of the BAC-latanoprost treated group in thisstudy, including decreases in basal tear secretion and goblet

FIGURE 3. Representative images of CIC on day 61 (HP, 3400). (A) In the control group, the epithelial cells (white arrow) were small and roundwith a nucleocytoplasmic ratio (NCR) of 1:2. Abundant goblet cells (black arrow) were plump, oval with intensely PAS-positive cytoplasm. The CICgrade was 0. (B) In the PBS-treated latanoprost group, the epithelial cells appeared enlarged with a NCR of 1:4 to 1:5 and pyknotic nuclei. The cell-to-cell junctions were loose and the cells appeared isolated. Goblet cells were smaller and markedly fewer, less intensely PAS-positive with poorlydefined cellular borders. The CIC grade was 2 . (C) In the SH-treated latanoprost group, most of epithelial cells were small and round with a NCR of1:2, a few epithelial cells appeared with an NCR of 1:3. Goblet cells were abundant, oval with intensely PAS-positive cytoplasm. The CIC grade was 0to approximately 1.

FIGURE 2. Alterations of aqueous tear production and conjunctival GCD in each group. (A) Compared with the control group, aqueous tearproduction decreased in the PBS-treated latanoprost group on days 31 and 61 (both P < 0.001). In the SH-treated group, aqueous tear productionwas significantly increased compared with the PBS-treated group on days 31 and 61 (both P < 0.01). (B) Compared with the control group, the PBS-treated (both P < 0.001) and SH-treated (both P < 0.05) latanoprost groups showed a trend of decreased GCD on days 31 and 61, but statisticallysignificant differences were found between the SH-treated and PBS-treated groups (P < 0.01, P < 0.001, respectively) on days 31 and 61.



cell density, increases in inflammatory cell infiltration, andapoptosis rates. It seemed that the protective effects oflatanoprost were not enough to neutralize the toxicities ofBAC.

BAC induces damage in the superficial structure andintegrity.26 Fluorescein staining means the disruption ofcorneal epithelial cell-cell junctions and damaged cornealepithelial cells.27 Rose bengal staining means deficiency in tearfilm (especially mucin layer), which covers corneal andconjunctival epithelial cells.28 The Schirmer test is the classicmethod for measuring the production of the aqueouscomponent in tears.26 Microvilli on the surfaces of cornealand conjunctival epithelial cells play a key role in theadherence of tear film mucin layer to epithelial cells. UnderSEM and TEM, ultrastructural disorders of the epithelial cellsand microvilli could be observed clearly. The results of ourstudy showed that SH could decrease fluorescein and rosebengal staining induced by application of BAC-preservedlatanoprost and could increase aqueous tear production andprotect ultrastructures, such as microvilli on the epithelialcells. A rational explanation may be that SH, a viscousbiopolymer with negative charges, can neutralize the toxiccationic charge of the remaining BAC quaternary ammonium.29

Moreover, viscoelastic properties and long ocular surfaceresidence time of SH have rendered it effectively increase tearfilm stability and corneal wettability, and reduce the tearevaporation rate.30 It was recently reported that a hyaluronatereceptor, CD44, is expressed in human and rabbit corneas.31,32

SH promoted human cornea epithelial cell migration by theadhesion between CD44 receptor.33

Dogan et al.7 found that the apoptosis rates of epithelialcells were higher in glaucoma patients; however, no differencewas found between different antiglaucoma medications. In areconstructed three-dimensional model of human cornealepithelial cells (3D-HCEs), BAC induced more positive TUNELstaining (representing late stage of apoptosis and cell death) inthe superficial cell layers and more activation of caspase-3(representing early stage of apoptosis) in the deeper layers.34

We also demonstrated that BAC-preserved latanoprost in-creased TUNEL staining especially in the superficial layer ofconjunctival epithelium; nevertheless, this phenomenon wassignificant suppressed by topical application of SH. Becauseprotection against ocular surface disorders could be achievedby suppression of apoptosis,35,36 we consider that SH couldeffectively protect the ocular surface during topical antiglau-comatous therapy.

FIGURE 4. Representative images of conjunctival paraffin sections with hematoxylin-eosin staining on day 61 in each group (HP, 3400). (A) Thecontrol group showed normal rabbit conjunctiva with few lymphocyte infiltration in the epithelium and stroma. (B) The PBS-treated latanoprostgroup showed remarkable inflammatory cell infiltration. (C) The SH-treated latanoprost group showed less inflammatory cells infiltration in theconjunctiva. (D) Alterations of the inflammatory cells count in the conjunctival epithelium and stroma in each group on day 61. Compared with thecontrol group, both PBS-treated (P < 0.001) and SH-treated (P < 0.05) latanoprost groups showed a trend of increased inflammatory cell infiltration.In the SH-treated group, conjunctival inflammatory cell infiltration was significantly less than the PBS-treated group (P < 0.001).



There are three possible mechanisms through which SH

decreases the apoptosis rate. First, BAC can induce reactive

oxygen species overproduction, which is associated with

alterations of the mitochondrial respiratory chain and results

in stretching of mitochondrial crests and formation of

megamitochondria.37 The mitochondrial dysfunction induces

the activation of apoptogenic proteases with secondary

endonuclease activation and consequent apoptosis.8 SH is rich

in hydroxyl functions, which may potentially absorb reactive

oxygen species induced by BAC. Second, several recent studies

indicated that BAC-induced P2X7 receptor activation could

cause cytolytic pore formation and lead to apoptosis.38 SH

physically coated cell membrane via strong links with CD44

receptors. At the same time, it masked the P2X7 receptors, and

inhibited the P2X7 receptors to be activated.15 Finally,

toxicological studies showed in conjunctival cell lines, the

prostaglandin analogue seemed to present cytoprotective and

antioxidative effects against BAC-induced toxicity.23–25 These

FIGURE 6. Representative images of SEM for the corneal epithelium on day 61. (A) the control group showed many bright cells with hexagonalshape ([A-1], SEM 33000), high-density microvilli ([A-2], SEM 324,000). (B) The PBS-treated latanoprost group showed diffuse cell peeling, loss ofhexagonal shape, and plasma cell wrinkling ([B-1], SEM 33000), diffuse loss of microvilli ([B-2], SEM 324,000). (C) The SH-treated latanoprost groupshowed mild cell peeling and slight cell wrinkling ([C-1], SEM 33000), and high density of microvilli ([C-2], SEM 324,000).

FIGURE 5. Representative images of the conjunctival TUNEL staining on day 61 (HP, 3400). (A) The control group showed few apoptotic cells in theepithelium. (B) The PBS-treated latanoprost group showed large amounts of apoptotic cells (brown) particularly among the epithelial and the gobletcells. (C) The SH-treated latanoprost group showed a significant drop in the number of apoptotic cells.



protective effects of latanoprost became possibly apparentafter BAC neutralization by SH. That is to say, we canhypothesize that SH and latanoprost play a synergic role inreducing BAC-induced toxicity.

A more severe side effect of BAC-preserved antiglaucomadrops is chronic ocular surface inflammation. Broadway andcolleagues39 found that subclinical inflammation induced bythe use of topical preserved antiglaucoma drugs was associatedwith a significantly lower trabeculectomy success rate incomparison with patients undergoing initial trabeculectomy.Baudouin et al.6 showed that glaucoma patients treated over 1year, even though clinically asymptomatic, exhibited signifi-cant expression of HLA-DR, ICAM-1, IL-6, IL-8, and IL-10 in theconjunctival epithelium. BAC-preserved drugs may causeapoptosis in epithelial cells, increased renewal of theconjunctival epithelium, release of inflammatory cytokines,and further activation of inflammatory cells. Our studydemonstrated that SH could effectively decrease inflammatorycell infiltration induced by BAC in the conjunctival epitheliumand stroma. Ex vivo and in vivo studies showed that BAC-preserved latanoprost induced fewer positive cells for inflam-matory marker expression compared with 0.02% BAC-pre-served timolol23 or 0.02% BAC alone.40 In this work, thelatanoprost anti-inflammatory effect observed may be promot-ed after BAC neutralization by SH. Besides the antioxidativenature and possible cooperation with latanoprost, SH maydecrease expression of apoptosis-related inflammatory mark-

ers, Fas, APO2.7, HLA-DR, and CD40 through CD44 mediation,just like in treatment of moderate dry eye syndrome andsuperficial keratitis.41 A limitation of this study is lack ofdetecting the expression of inflammatory cytokines. Ourfurther understanding of the mechanism was limited due tothe poor availability of antibodies against rabbit proteins.Further studies are required.

GCD is a key parameter that reflects the overall health ofthe ocular surface.42 Conjunctival impression cytology is themost effective noninvasive technique for counting goblet cells.Current studies showed that BAC might trigger a direct toxicityon goblet cells and inhibit mucin production in goblet cells.26

Pisella et al.23 found that in patients treated with BAC-preserved antiglaucoma drugs, the lowest levels of MUC5AC-positive cells were related to the highest HLA-DR and ICAM-1levels, thus emphasizing that inflammation in conjunctiva wasassociated with a highly significant decrease in goblet cells.Inflammatory immune environment caused apoptosis of gobletcells and decreased production of secretory granules, resultingin an unstable tear film, thereby aggravated ocular surfacedamage and stimulated the inflammation cascade of theepithelial cells. Thus, the eye entered a vicious cycle.43 Inour study, SH significantly improved GCD and conjunctivalsquamous metaplasia. Moreover, TEM also showed moreglycogen inside goblet cells in the SH-treated latanoprost thanin the PBS-treated group. SH possibly maintains goblet celldensity and stimulates mucin synthesis by neutralizing BAC and

FIGURE 8. Representative images of TEM for the conjunctival epithelium on day 61 (TEM, 36000). (A) the control group showed abundant Mv onthe conjunctival epithelium and secretary granules (Sg) that store mucins in the goblet cells. (B) The PBS-treated latanoprost group showeddisruption of the Mv and reduction of Sg. (C) The SH-treated latanoprost group showed abundant Mv on the conjunctival epithelium and Sg in thegoblet cells.

FIGURE 7. Representative images of TEM for the corneal epithelial cells on day 61 (TEM, 315,000). (A) The control group showed high-densitymicrovilli (Mv), nuclei (Nu) with evenly distributed chromatin, abundant organelles such as mitochondria (Mi), Golgi apparatus (Go), and roughendoplasmic reticulum (RER). (B) The PBS-treated latanoprost group showed disruption of the Mv, swelling of Mi and RER, granular clumps in thenuclear chromatin, and widening of the perinuclear cisterna (PN). (C) The SH-treated latanoprost group showed nuclei (Nu) with evenly distributedchromatin, and abundant Mv, Mi, Go, and RER.



also through its antioxidative and anti-inflammatory properties,which might help the ocular epithelium break out of thevicious cycle of apoptosis, inflammation, and goblet cell loss.

The model consisting of regular applications that mimic theclinical setting in rabbit eyes does not reflect the real ocularsurface reactions in patients, even rabbit corneal tissue appearsto be more sensitive to the exposure of antiglaucomamedications than human tissues.5 But it may emphasize theactions of SH and the histomorphological changes that aredifficult to obtain from patients.

In conclusion, our in vivo study displayed that SHsignificantly reduced the ocular surface toxicity induced byone commonly used antiglaucoma medication, preservedlatanoprost. As a vehicle or neutralizing material, SH couldbe proposed to reduce ocular toxicity, and protect the ocularsurface in long-term BAC-preserved latanoprost treatment.

Acknowledgments

Supported by the International Scientific and TechnologicalCooperation Projects of Guangdong Province, China (Grant2012B050600032). There were no financial, personal, or profes-sional interests that could be construed to have influenced thewriting of this paper.

Disclosure: F. Yu, None; X. Liu, None; Y. Zhong, None; X. Guo,None; M. Li, None; Z. Mao, None; H. Xiao, None; S. Yang, None

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