Olopatadine enhances recovery of alkali-induced corneal

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Life Sciences

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Olopatadine enhances recovery of alkali-induced corneal injury in rats

Samah Kandeela,⁎, Mohamed Balahab

aHistology Department, Faculty of Medicine, Tanta University, El-Gish Street, Postal No. 31527 Tanta, Egyptb Pharmacology Department, Faculty of Medicine, Tanta University, El-Gish Street, Postal No. 31527 Tanta, Egypt

A R T I C L E I N F O

Keywords:CorneaAlkaliIL-1βVEGFCaspase-3NF-κB expression

A B S T R A C T

Aims: The alkali-induced corneal injury is an ocular emergency that required an immediate and effectivemanagement to preserve the normal corneal functions and transparency. Olopatadine is a fast, topically-effectiveanti-allergic drug, which exhibited potent anti-inflammatory and anti-angiogenic abilities in different allergicanimals' models. Therefore, this study aimed to evaluate the therapeutic effect of olopatadine on alkali-inducedcorneal injury in rats.Materials and methods: Corneal alkali injury (CI) induced in the right eyes of an eight-week-old male Wister rats,by application of 3mm diameter filter-papers, soaked for 10 s in 1 N-NaOH, to the right eyes' corneal centers for30 s, afterward, the filter paper removed, and the rat right eye rinsed with 20ml normal saline. For treatment ofCI, either 0.2% or 0.77% olopatadine applied topically daily for 14 days, starting immediately after the inductionof CI.Key findings: Olopatadine, in the present work, effectively and dose-dependently enhanced the corneal healingafter alkali application, with significant reduction of the corneal opacity and neovascularization scores, besides,it suppressed the augmented corneal IL-1β, VEGF, caspase-3 levels, and nuclear NF-κB immunohistochemicalexpression, meanwhile it abrogated the corneal histopathological changes, induced by alkali application.Significance: Olopatadine appears to be a potential treatment option for alkali-induced corneal injury.

1. Introduction

Chemical injuries of the eye account for 11.5–22.1% of all theocular traumas, where two-thirds of which affecting young men. Mostof these injuries occur as a result of industrial accidents in agriculture,laboratories, fabric mills and automotive repair facilities, while theminority occurs at home, and as a secondary assault. The alkali-inducedocular injury is account nearly for 60% of all chemical ocular injury,and considered as an ocular emergency, where immediate and effectivemanagement is needed to preserve the normal corneal functions andtransparency, otherwise, severe corneal injury, with slow re-epitheli-zation, ulceration, neovascularization, and opacification will be settled,with subsequent corneal and anterior chamber destruction that endsultimately into loss of vision [1–4]. The common alkali that causescorneal injury are lye, ammonia, potassium and magnesium hydroxide,and lime, which present in many cleaning agents, drain cleaners,building material, staining dyes, fertilizer, laboratory reagents, andfirework sparklers, where, lime is considered the most common agent,and ammonia and lye, which contains NaOH, are the most dangerousones [3, 4].

Furthermore, alkali agents are lipophilic in nature, thus can

penetrate tissues very rapidly, with consequent saponification the cellmembranes' fatty acids [5]. Moreover, the hydroxyl group present inmost alkalis penetrates the corneal stroma, destroying its proteoglycanground substance, as well as the collagen bundles, with subsequentproduction of proteolytic enzymes from the damaged tissue, as a con-sequence of the inflammatory response, leading to further chemicalpenetration [2, 6–8]. Additionally, the exposure of the eye to alkaliaffect the ocular surface epithelium, in addition to its adnexa, withspecial effect on the cornea leading to its apoptosis, inflammation,perforation, neovascularization, and opacification, with recruitment ofinflammatory cells, mesenchymal cells, activated keratocytes andmacrophages that orchestrating the processes of corneal injury, repair,and wound healing, through the release of various cytokine, angiogenicand anti-angiogenic factors [7, 9]. Where, the imbalance between theangiogenic and anti-angiogenic factors, with predominance of angio-genic factors such as vascular endothelial growth factor (VEGF), andthe increased release of the pro-inflammatory cytokines, such as in-terleukin (IL)-1β, with activation of caspase and nuclear factor-kappa B(NF-κB) cascades are thought to be the main pathogenesis of cornealopacity and neovascularization, following the alkali injury [9–11].Accordingly, by a proper understanding of the alkali corneal injury's

https://doi.org/10.1016/j.lfs.2018.07.002Received 23 April 2018; Received in revised form 30 June 2018; Accepted 2 July 2018

⁎ Corresponding author.E-mail address: [email protected] (S. Kandeel).

Life Sciences 207 (2018) 499–507

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pathogenesis, new treatment modalities can be assigned for appropriatemanagement of corneal alkali injury to prevent its sequelae, as well as,to preserve the normal corneal functions and transparency.

At present, most of the therapeutic modalities used for the treatmentof alkali-induced corneal injuries, inflammation and inflammatoryneovascularization, are less effective in restoration of normal cornealfunctions and transparency [1, 9]. As, most of the medications used,including irrigations, steroids, citrate, medroxyprogesterone, non-ster-oidal anti-inflammatory drugs, hyaluronate, artificial tears, fibronectin,retinoic acid, ascorbate, tetracyclines, macrolides, and fluor-oquinolones, have many adverse effects, and its clinical outcomes arenot satisfactory [9, 12]. Moreover, Bakunowicz-Łazarczyk and Urban[9], Sharma et al. [1] and Singh et al. [2] reported that the surgicalintervention of the corneal injury by debridement of corneal necroticepithelium is associated with the release of proteolytic enzymes, withfurther aggravating of the corneal damage. As well, amniotic membranetransplantation, limbal stem cell transplant, cultivated oral mucosalepithelial transplantation, or corneal transplantation complicated withsymblepharon formation, corneal neovascularization, limbal stem celldeficiency, and high rate of graft rejection. Additionally, Trief andColby [3] documented that whatever the healthy appearance of the eyeafter surgical intervention, the patient should be followed up as15–50% of them usually complicated by glaucoma and dry eye syn-drome. Consequently, new approaches are needed to improve themanagement of corneal alkali injury as regards for the inflammatory,apoptotic and angiogenesis-related factors.

Olopatadine is a fast topically effective anti-allergic drug, used inthe treatment of allergic conjunctivitis, rhinitis, urticaria, dermatitis,pruritus, and erythema exudative multiform [13–15]. It is a potent dualacting mast cell stabilizer, and selective histamine-1 receptor antago-nist, so, it inhibits type 1 immediate hypersensitivity reaction, and re-strains the release of mast cell inflammatory mediators like histamine,tryptase and prostaglandin D2, with subsequent suppression of in-flammatory and pro-inflammatory cytokine release, and inflammatorycells and conjunctival epithelium recruitment [13, 16–18]. Further-more, it exhibited potent anti-inflammatory, anti-edematous and anti-angiogenic abilities in a mice and rat models of allergic conjunctivitisand rhinitis respectively, through the suppression of the pro-in-flammatory and inflammatory cytokine, such as IL-1β, and the angio-genic factors, such as VEGF, production [14, 19]. Additionally, topicalocular application of olopatadine as a single daily dose documentedclinically to be effective and safe for long-term use in the treatment ofallergic conjunctivitis [13, 18, 20, 21]. Therefore, this study aimed toevaluate the therapeutic effect of olopatadine on the corneal opacityand neovascularization, through the assessment of its anti-in-flammatory, anti-angiogenic and anti-apoptotic activities, as well as itsabrogative potentiality on the corneal histopathological changes andimmunohistochemical NF-κβ expression, in a rat model of alkali-in-duced corneal injury.

2. Materials and methods

2.1. Drugs and chemicals

Olopatadine hydrochloride and diaminobenzidine purchased fromSigma, St. Louis, MO, USA, ketamine from Sigma, Nasr City, Cairo,Egypt, xylazine from Adwia, Obour City, Cairo, Egypt, hematoxylinstain from Oxford, India, eosin stain from Jahangir, India, and sodiumpentobarbitone from Abbott Lab., Chicago, IL, USA. Moreover, phos-phate-buffered saline (PBS), Tris-HCl buffer, formalin-buffered saline,Canada balsam, periodic acid, Schiff's reagent, hydrogen peroxide,methanol and sodium hydroxide (NaOH) obtained from El GomhuriaCo., Tanta, El-Gharbeya, Egypt. However, serum-free protein blockingsolution (Block Ace) bought from DS Pharma Biomedical Co., Ltd.,Osaka, Japan and normal saline from Otsuka Egypt Co., Nasr City,Cairo, Egypt.

2.2. Animals

Eight-week-old male Wister rats, weighing 150–200 g, obtainedfrom Tanta Faculty of Medicine animal house, Egypt, used in the pre-sent study. Rats housed in meshed plastic cages under standard la-boratory conditions, with free access to standard laboratory food andwater Ad libitum. Animals permitted to adapt for one week before ex-perimentation start. All animals' managements followed the Associationfor Research in Vision and Ophthalmology (ARVO) animals' guidelines,which were in accordance with Tanta Faculty of Medicine guidelinesfor care and use of Animals in research, with the approval of TantaFaculty of Medicine's Animal Experiment Ethics Committee, Egypt.

2.3. Induction of alkali corneal injury

Alkali corneal injury induced in the right eyes of rats only, withsparing of the left one to ensure the animals welfare. It induced ac-cording to Arikan et al. [22], briefly, 3mm diameter filter paper soakedin 1 N NaOH for 10 s (sec), then the excess NaOH removed, and appliedto the corneal center of the rat right eye for 30 s, after the anesthesia ofrat with intraperitoneal injection of 75mg/kg ketamine and 5mg/kgxylazine. Later, the filter paper removed, and the rat right eye rinsedwith 20ml normal saline. Whereas, normal saline instead of NaOHapplied to the rats' right eyes of the control group [22].

2.4. Experimental design

Sixty Wister rats randomly distributed into 5 groups of 12 rats each.Group I (CON), was normal rats. Group II (CI), was NaOH-inducedcorneal injury rats. Group III (VEH), was corneal injury induced rats,treated topically with 10 μl/eye/day PBS. Group IV (OL), was cornealinjury induced rats, treated topically with 10 μl/eye/day olopatadine0.2% (dissolved in PBS) [23]. Group V (OH) was corneal injury inducedrats, treated topically with 10 μl/eye/day olopatadine 0.77% (dissolvedin PBS) [23]. All treatments were freshly prepared daily and appliedonce daily for 14 days between 10:00 A.M. and 12 P.M., starting im-mediately after the induction of corneal injury.

2.5. Corneal opacity and neovascularization scores

The corneal opacity and neovascularization scores assessed at 3rd,7th, 10th and 14th day of treatment, after the application of NaOH, byan ophthalmoscope (HS-OP10, Belson, China). The corneal opacityscore determined with the method described by Ke et al. Where, grade0, normal transparent cornea, grade 1, minimal corneal opacity, grade2, mild corneal opacity with pupil margin and iris vessels were visible,grade 3, mild corneal opacity with only pupil margin was visible, grade4, moderate corneal opacity with only pupil still visible, grade 5,complete corneal opacity [24]. However, the corneal neovasculariza-tion score evaluated according to the method reported by Dana andStreilein. Shortly, the cornea divided into 4 quadrants, then neo-vascularization of each quadrant graded between 0 and 3, according tothe neovascular branch centripetal extension from the corneoscleraljunction. Where, grade 0, no neovascularization, grade 1, the neo-vascularization< 1/3 of corneal radius, grade 2, neovascularizationbetween 1/3 and 2/3 of corneal radius, grade 3 the neovasculariza-tion> 2/3 of the corneal radius. Afterward, the grade of all quadrantssummed to get 0 to 12 corneal neovascularization score for each eye[25]. Both corneal opacity and neovascularization scores expressed asarbitrary units.

2.6. Samples collection

Twenty-four hours after the last treatment, rats sacrificed by cer-vical dislocation. Then the rats' right eyes dissected and their corneaharvested. The corneas of six rats per each group immediately

S. Kandeel, M. Balaha Life Sciences 207 (2018) 499–507

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processed for histopathological and immunohistochemical evaluation.However, each cornea of the other six, instantly homogenized in 1ml of0.1 mmol Tris-HCl buffer (pH=7), and centrifuged at 5000 rpm for10min, afterward, the supernatant collected and stored at −80 °C forfurther assessment.

2.7. Assessment of corneal IL-1β level

The corneal IL-1β level estimated according to the manufacturer'sinstruction of an enzyme-linked immunosorbent assay (ELISA) kitpurchased from Bio Vendor Laboratorní medicína A.S., Karasek, CzechRepublic, with a minimum detection limit of 4 pg/ml. The absorbanceof the samples determined using an automated ELISA plate reader (StatFax 2100, Fisher Bioblock Scientific, BP., Illkirch Cedex, France), andthe corneal IL-1β level expressed as pg/ml.

2.8. Assessment of corneal VEGF level

The corneal VEGF level determined with an ELISA kit obtained fromCUSABIO, Donghu, Hi-Tech, Wuhan, Hubei Province, China, with aminimum detection limit of 0.97 pg/ml, as described by its producer.The samples' optical densities analyzed with an automated ELISA platereader (Stat Fax 2100, Fisher Bioblock Scientific, BP., Illkirch Cedex,France), and the corneal VEGF level expressed as pg/ml.

2.9. Assessment of corneal caspase-3 level

An ELISA kit acquired from Sunred Bio. Tec., Shangai, China, withminimum detection limit 0.045 ng/ml used to estimate the cornealcaspase-3 level, according to the manufacturer's protocol. The samples'absorbance assessed by an automated ELISA plate reader (Stat Fax2100, Fisher Bioblock Scientific, BP., Illkirch Cedex, France), and thecorneal caspase-3 level expressed as ng/ml.

2.10. Assessment of corneal histopathological changes

Corneal samples prepared according to [26]. Concisely, specimenstook form 6 rats per each group and directly fixed in 10% formalin-buffered saline. Then, dehydrated, cleared and impregnated into par-affin. Finally, a rotatory microtome (Olympus, Tokyo, Japan) used toobtain sections of 5 μm, used for the histological and im-munohistochemical study [26].

2.10.1. Hematoxylin and eosin (H & E) stainingCorneal specimens' sections deparaffinized, hydrated and stained

with hematoxylin for 30min (min), then with 1% eosin for 10min.Finally, sections dehydrated, cleared, mounted in Canada balsam andexamined with a light microscope (Olympus, Tokyo, Japan).

2.10.2. Periodic acid Schiff's (PAS) stainingSpecimen's deparaffinized and hydrated, then, immersed in 1%

periodic acid followed by Schiff's reagent for 15min, after that dehy-drated, cleared and mounted in Canada balsam. Lastly, magenta colorstaining of basement membranes saw using a light microscope(Olympus, Tokyo, Japan).

2.10.3. Immunohistochemical localization of the NF-κB-p65In short, sections deparaffinized, rehydrated, then, the endogenous

hydrogen-peroxidase activity blocked by placing sections in 0.3% hy-drogen peroxide/methanol for 20min, then, washed with PBS.Afterward, a serum-free protein blocking solution (Block Ace) added tothe sections and incubated for 20min, later, the sections incubated withthe rabbit anti-NF-kB-p65 antibody (1:200, Santa Cruz Biotechnology,California, U.S.A.) overnight at 4 °C. Next, sections washed with PBSand incubated with the secondary antibody (Dako North America, Inc.,CA, USA) for 10min. Finally, 1-2drops of diaminobenzidine added to

each slide for 5–10min, then, counterstained with hematoxylin, dehy-drated, cleared and examined by a light microscope (Olympus, Tokyo,Japan). Nuclear and cytoplasmic brown reaction got. The negativecontrol specimens obtained through the addition of PBS rather than theprimary antibody [27]. Furthermore, the nuclear and cytoplasmic NF-κB-p65 expression each alone arbitrary scored according to Guo et al.[28]. In brief, the percentage of cells that expressed a positive reactionin either cytoplasm or nucleus each alone determined per 200 cells in×400 magnified images per each rat, and scored arbitrary, where, 0,0–9%, 1, 10–25%, 2, 26–50% and 3,> 50% of cells expressed a positivereaction. Moreover, the positive reaction intensity, in the same cells,scored arbitrarily as follow, 0, no reaction, 1, weak reaction, 2, mod-erate reaction and 3, strong reaction. Then, the total im-munohistochemical score was estimated by the following formula“percentage of positive reaction cells score X positive reaction intensityscore”, where the score ranged from 0 to 9 arbitrary units, for the nu-clear and cytoplasmic NF-κB-p65 expression each alone [28].

2.11. Statistical analysis

The data of the present study expressed as mean ± SD. The sta-tistical difference between the different groups evaluated by one-wayANOVA (followed by Tukey's test as a post hoc test) or Kruskal-Wallis'stest (followed by Mann-Whitney's U test as a post hoc test), after ana-lysis of variances with Bartlett's test. However, the corneal opacity andneovascularization scores' data analyzed by two-way ANOVA, followedby Tukey's test as a post hoc test. The significance of data consideredwhen the P value < 0.05.

3. Results

3.1. Effect of olopatadine on the corneal opacity and neovascularizationscores

Alkali application significantly induced a severe corneal injury,which could be revealed by a coarse corneal opacity that deepened withtime. Furthermore, the healing process, after NaOH application, asso-ciated with a progressive growth of neovascular branches from thecorneoscleral junction toward the corneal center. In contrary, the dailytopical treatment with olopatadine effectively and dose-dependentlyenhanced the corneal healing after alkali application, with a significantreduction of the corneal opacity and neovascularization scores, whencompared with CI group. Additionally, the reduction of the cornealopacity by olopatadine showed no significant difference throughout thetime course of its assessment, in contrary to the neovascularizationscores where there is a significant increase of it by the 14th day of theexperiment, when compared with the 3rd day (Fig. 1).

3.2. Effect of olopatadine on the corneal IL-1β level

In parallel with the severe corneal opacity and neovascularizationthat induced by NaOH application, the corneal IL-1β level significantlyaugmented after alkali application, as compared to the control group.The daily treatment with topical olopatadine efficiently suppressed theamplified corneal IL-1β level, induced by alkali application, in a dose-dependent manner (Fig. 2).

3.3. Effect of olopatadine on corneal VEGF level

In correspondent to the neovascularization score results, the cornealVEGF level significantly enhanced, in the corneal injury induced byalkali application, when compared to the control group. However, thedaily treatment with topical olopatadine application, after alkali-in-duced corneal injury, powerfully suppressed the enhanced cornealVEGF level dose-dependently (Fig. 2).


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3.4. Effect of olopatadine on the corneal caspase-3 level

The corneal apoptosis significantly increased by NaOH topical ap-plication, as indicated by significant elevation of the corneal caspase-3level, when compared with the caspase-3 level of the control group. Thedaily topical application of olopatadine, for treatment of the cornealalkali injury, effectively and dose-dependently reduced the elevatedcorneal caspase-3 level induced by alkali injury. Furthermore, the OHgroup showed a significant elevation of the caspase-3 level, whencompared with the control group (Fig. 3).

3.5. Effect of Olopatadine on corneal histopathological changes

3.5.1. H & E stained sectionsCorneal H & E stained sections of the control group showed five

distinct layers arranged from outside inward as follow, the 1st layer wasthe corneal epithelium that was non-keratinized stratified squamousepithelium resting on a basement membrane, and consisting of basalcolumnar cells with oval basal nuclei, however, the 2nd layer consistedof two to three layers of intermediate polygonal cells, with roundednuclei, and one or two layers of superficial squamous cells, with flat-tened nuclei, moreover, the 3rd layer was a cellular homogenousBowman's membrane, in addition to the 4th layer, which was stroma or

Fig. 1. Effect of olopatadine on the corneal opacity and neovascularization scores in male Wister rats' alkali-induced corneal injury. A) Results of corneal opacity andneovascularization scores, expressed as mean ± SD of 6 rats per each group. CON, normal rats, CI, NaOH-induced corneal injury rats, VEH, corneal injury inducedrats, treated topically with 10 μl/eye/day PBS, OL, corneal injury induced rats, treated topically with 10 μl/eye/day olopatadine 0.2%, OH, corneal injury inducedrats, treated topically with 10 μl/eye/day olopatadine 0.77%, and N, normal cornea. *P < 0.05 (Vs. CI group), #P < 0.05 (Vs. OL group), a P < 0.05 (Vs. OL groupat 3rd day) and b P < 0.05 (Vs. OH group at 3rd day). B) Representative images of corneal opacity and neovascularization in different groups.

Fig. 2. Effect of olopatadine on the corneal IL-1β and VEGF levels in male Wister rats' alkali-induced corneal injury. Results expressed as mean ± SD of 6 rats pereach group. CON, normal rats, CI, NaOH-induced corneal injury rats, VEH, corneal injury induced rats, treated topically with 10 μl/eye/day PBS, OL, corneal injuryinduced rats, treated topically with 10 μl/eye/day olopatadine 0.2%, and OH, corneal injury induced rats, treated topically with 10 μl/eye/day olopatadine 0.77%.**P < 0.01 & ***P < 0.001 (Vs. CI group) and ###P < 0.001 (Vs. OL group).


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substantia propria that appeared as avascular parallel lamellae of densecollagenous fibers, with flattened fibroblasts (keratocytes) scattered in-between them, the last layer was flattened endothelial cells that linedthe inner surface of the cornea, and supported by Descemet's membranethat appeared as a thick homogenous basement membrane. The ex-amination of CI & VEH groups' sections revealed a severe corneal injurythat evidenced by the presence of deeply stained epithelial cells' nucleithat surrounded by vacuolated cytoplasm, with irregularities ofBowman's membrane. Moreover, the stroma exhibited areas of widelyseparated collagen lamellae, as well as mononuclear cellular infiltra-tion, and stromal vascularization. Furthermore, some of the endothelialcells appeared swollen with some focal parts stratified, with focal se-paration from the Descemet's membrane. Conversely, OL & OH groupsrevealed nearly restoration of the normal corneal histological structure

dose-dependently (Fig. 4).

3.5.2. PAS-stained sectionsThe PAS-stained sections of the control group showed a PAS-posi-

tive magenta colored epithelial basement membrane and Descemet'smembrane. Whereas the CI &VEH groups revealed focal interruption ofthe epithelial basement membrane, that proved by the interruption oreven absence of the PAS-positive reaction in some areas, while, theDescemet's membrane displayed a normal PAS positive reaction. On theother hand, the OL & OH groups demonstrated a dose-dependent en-hancement of PAS-positive reaction of the epithelial basement mem-brane to be apparently like the control group in the high-dose treatedolopatadine group (Fig. 5).

Fig. 3. Effect of olopatadine on the corneal caspase-3 level, and corneal epithelial NF-κB-p65 immunohistochemical expression in male Wister rats' alkali-inducedcorneal injury. Results expressed as mean ± SD of 6 rats per each group. CON, normal rats, CI, NaOH-induced corneal injury rats, VEH, corneal injury induced rats,treated topically with 10 μl/eye/day PBS, OL, corneal injury induced rats, treated topically with 10 μl/eye/day olopatadine 0.2%, OH, corneal injury induced rats,treated topically with 10 μl/eye/day olopatadine 0.77%, and ND, not detected. ***P < 0.001 (Vs. CI group), ##P < 0.01 & ###P < 0.001 (Vs. OL group) and @P < 0.05 (Vs. OH group).

Fig. 4. Effect of olopatadine on the corneal histopathological changes in H & E stained sections of male Wister rats' alkali-induced corneal injury. A, CON group,showed five distinct layers, non-keratinized stratified squamous epithelium resting on a basement membrane, and consisting of basal columnar cells with oval basalnuclei (1), two to three layers of intermediate polygonal cells with rounded nuclei (2) and one or two layers of superficial squamous cells with flattened nuclei (3),Bowman's membrane appeared homogenous and a cellular (→), avascular stroma with parallel lamellae of dense collagenous fibers and flattened fibroblasts(keratocytes) (*), Descemet's membrane that appeared as a thick homogenous basement membrane (►), flattened endothelial cell lined the inner surface of thecornea (curved arrow). B1, CI group, showed deeply stained epithelial cell nuclei, with vacuolated cytoplasm (→), stroma with widely separated collagen lamellae(*), mononuclear cellular infiltration (two arrows), stratification of endothelial cells (►), with focal separation from the Descemet's membrane (curved arrow). B2, CIgroup, with Bowman's membrane irregularities (→), and stromal vascularization (two arrows). C, VEH group, showed deeply stained epithelial cell nuclei, withvacuolated cytoplasm (→), stroma with separated collagen lamellae (*), mononuclear cellular infiltration (two arrows), and some of the endothelial cells appearedswollen (►). D, OL group, revealed stromal irregularity (*), and vascularization (→). E, OH group, showed nearly normal corneal epithelium (two arrows), Bowman'smembrane (→), stroma with fibroblasts (*), Descemet's membrane (►) and endothelial layer (curved arrow). (H & E. Scale bar= 50 μm).


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3.6. Effect of olopatadine on the corneal epithelial NF-κB-p65immunohistochemical expression

Corneal epithelial NF-κB-p65 immunohistochemical expressionshowed different localizations in the experimental groups, by which it iscytoplasmic in the control group, while, in CI & VEH groups, it becamenuclear because of increased NF-κB-p65 nuclear translocation.Moreover, in olopatadine treated groups, there is a significant and dose-dependent elevation of cytoplasmic and reduction of nuclear NF-κB-p65immunohistochemical expression, when compared to CI group.Nevertheless, the OH group showed a significant reduction of cyto-plasmic and significant elevation of nuclear NF-κB-p65 im-munohistochemical expression, when compared with the control group(Figs. 3 & 6).

4. Discussion

The cornea is an avascular transparent part of the eye that performsan important role in the maintenance of clear eye vision and is con-sidered as a notable immune site in the body. Moreover, the cornealepithelium considered as the first defensive line of the eye against pa-thogen-associated molecular pattern recognition that initiates stronginnate immune responses. The exposure of the eye to the alkali, inducea chronic immune-mediated inflammation, with subsequent disruptionsof the ocular surface structure and function [29, 30].

Our model in the present study is a complete corneal alkali-burn,being used for assessment of the new medication effectiveness [22], asthe currently used therapeutics for corneal injuries are disappointing,insufficient, and not so effective in restoration of normal cornealfunctions and transparency. Moreover, most of the medications usedhave many adverse effects, and its clinical outcomes are not satisfac-tory. Furthermore, therapies that rapidly and effectively suppress in-flammation and angiogenesis are critical for the treatment of cornealchemical burns [1, 9, 12, 31].

The present work showed that alkali application was significantlyinduced a coarse corneal opacity that deepened with time and asso-ciated with a progressive evolution of neovascular branches from the

corneoscleral junction toward the corneal center. In addition, it sig-nificantly elevated corneal IL-1β, VEGF and caspase-3 levels as com-pared to the control group. Furthermore, it altered the histologicalstructure of the cornea, with widely separated collagen lamellae,stromal mononuclear cellular infiltrations, and neovascularization in H&E stained sections, and focal interruption of corneal epithelial base-ment membrane in PAS-stained section. In parallel with these finding,the corneal cytoplasmic and nuclear NF-κB immunohistochemical ex-pression significantly reduced and elevated by NaOH application to thecornea, respectively. In contrary, olopatadine in the present study dose-dependently abrogated these changes.

These previous findings can be explained by the fact that the ap-plication of alkali to the cornea induces corneal cauterization, withprotein denaturation, and dysregulation of cellular fluid homeostasis.Additionally, it promotes the local corneal inflammation, with re-quirement of inflammatory cells, mesenchymal cells, activated kerato-cytes and macrophages, which secrete local chemokines, pro-in-flammatory cytokine, such as IL-1β, and angiogenic factors, such asVEGF, also, it induced corneal cell apoptosis, with subsequent cellsdeath, corneal opacification and neovascularization [5, 11, 32]. More-over, the induced saponification creates a softened or liquid environ-ment that disrupts the cell membrane, and consequently, enables thealkali to spread over and over with impairment of the adjacent andinner tissues [3, 29].

Additionally, as a result of corneal epithelial insult, the damagedepithelium and its interrupted basement membrane, secrete pro-in-flammatory cytokines, mainly IL-1β, which stimulate corneal stromalkeratocyte to release Fas ligand that induces apoptosis of the nearbykeratocytes, thus augment IL-β secretion, with subsequent stromal re-quirement of macrophages, T-lymphocytes and neutrophils, whichphagocytosed the damage cell, and myofibroblast aiming to healing ofthe corneal injury [33]. Excessive secretion of IL-1β in corneal alkaliinjury promotes myofibroblast activity that enhances its laying-down offibrous tissue, with consequent corneal stromal remolding, haziness andopacification, besides, it enhance neutrophils recruitment that act inautocrine fashion secreting IL-1β, which initiates corneal angiogenesis,through the release VEGF, and reduces corneal healing rate, resulting

Fig. 5. Effect of olopatadine on the corneal histopathological changes in PAS-stained sections of male Wister rats' alkali-induced corneal injury. A, CON group,showed epithelial basement membrane (→), and Descemet's membrane (►) with PAS-positive magenta coloration. B, CI group, showed epithelial basementmembrane with interrupted even absent PAS+ ve reaction in some areas (→), and Descemet's membrane with normal PAS positive reaction (►). C, VEH group,revealed epithelial basement membrane with interrupted or even absent PAS+ ve reaction in some areas (→), and the Descemet's membrane with normal PASpositive reaction (►). D, OL group, with areas of focal loss and localized reduction of basement membrane PAS reaction (→). E, OH group, showed epithelialbasement membrane (→), and Descemet's membrane (►) with a PAS-positive reaction similar to control. (PAS. Scale bar= 50 μm). (For interpretation of thereferences to color in this figure legend, the reader is referred to the web version of this article.)


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into corneal damage [34–37]. Furthermore, IL-1β enhances expressionand activation of NF-κB-p65, with further amplification of in-flammatory cascades, hence, corneal damage, opacification, and neo-vascularization [11, 38, 39]. Also, IL-1β reported triggering cornealneovascularization directly, independent of angiogenic factors, such asVEGF, via enhancement of vascular endothelial cells migration andcapillary tubes development [40]. Additionally, treating of corneal al-kali injury with IL-1 receptor antagonist suppresses corneal inflamma-tion, with the promotion of corneal healing, re-epithelization, thus,restoration of its transparency [41].

Moreover, corneal cells alkali insult induces excessive expressionand secretion of VEGF, promoted by IL-1β, which in turn stimulatesVEGF receptor that mediates activation of phosphatidylinositol-3-ki-nase/protein kinase b, extracellular signal-regulated kinase/mitogen-activated protein kinase and protein kinase A/endothelial nitric oxidesynthase activation, with subsequent promotion of growth, survival,differentiation, proliferation and migration of the endothelial cells, aswell as, capillary tube formation, thus considered as the major mod-ulator of corneal wound healing and neovascularization [42, 43]. Also,the exposure of the eye to alkali cause corneal hypoxia, with subsequentpoor nutrition, as well as poor regeneration of the corneal epitheliumresults in corneal opacity, besides, it enhances the production of hy-poxia-inducible factor 1-alpha, which accumulates in the nucleus, withconsequent promotion of VEGF transcription and translation, thus,enhances corneal angiogenesis, that plays a fundamental role in the latecorneal healing stage, however the tremendous angiogenesis inducedby inflammatory reaction in alkali application results into cornealscarring, with subsequent corneal opacification and neovascularization[5, 19, 31, 44]. Furthermore, VEGF plays a pivotal role in the devel-opment of corneal inflammation following alkali injury, through thepromotion of macrophages corneal recruitment, thus initiates and/oraugments signals essential for corneal pathological neovascularization([40, 45]. Additionally, Choi et al. [6] added that neovascularization in-turn contributes to further opacification, through disruption of theregular collagen fibers, and edema because of vascular extravasation[6].

However, the present work revealed that olopatadine is effectivelyand dose-dependently enhanced the corneal healing after alkali appli-cation, with a significant reduction of the corneal opacity and

neovascularization scores, as well as suppression of the augmentedcorneal IL-1β and VEGF levels, besides, restoration of the normal cor-neal histological structure when compared with CI group. These find-ings are parallel with earlier studies, which proved that the treatmentwith olopatadine reduces the inflammatory reactions, as well as the IL-1β and VEGF levels in antigen-induced allergic conjunctivitis and rhi-nitis in mice and rats [14, 19, 46]. In addition, olopatadine has a sup-pressor effect on several inflammatory cells as it affects human eosi-nophils and neutrophils, with inhibition of ionophore A23187-inducedrelease of peptide-leukotrienes, suppression of thromboxane B2, andleukotriene B4 release from human eosinophils and neutrophils [47].Olopatadine, also, is a potent mast cell stabilizer, as well as anti-histaminic drug, as it inhibits the release of histamine and pros-taglandins from mast cells through the inhibition of mast cell Ca+2

influx, which is important for mast cell lysosomal exocytosis, conse-quently, inhibits the induction of histamine, with subsequent inhibitionof recruitment and activation of T lymphocytes, hence suppresses thesecretion of the pro-inflammatory mediators, such as IL-1β, and theangiogenic factors, such as VEGF [17, 48]. Moreover, the pro-in-flammatory cytokine, IL-1β, increase VEGF expression, then, VEGF in-duces a number of pro-inflammatory genes that in turn promotes trans-endothelial migration of neutrophils, therefore, the inhibition of theirsecretion by olopatadine, attenuated the neutrophil-mediated in-flammatory responses, with subsequent improving the corneal structureand function [49]. Nevertheless, the significant increase of the neo-vascularization score by the end of the 14th day, as compared to the3rd-day score, could be clarified as that the summit of the expressedmolecules in response to alkali injury is the 14th day after the injury,then its levels fad later on [37, 50, 51].

Furthermore, caspases are a family of important signaling mole-cules, when activated, multiple cellular damages settled. Caspase-3 isan executioner pro-enzyme, activated by caspase-8 and caspase-9, whenactivated, it initiates the “death cascade”, and so, it is considered as animportant marker of the apoptotic signaling pathway that leads tomorphological cell changes ending with apoptosis [52, 53]. In alkali-induced corneal injury, caspases play pleiotropic role in induction ofcorneal cells apoptosis, as well as, in induction of inflammatory cas-cades, through upregulation and activation of NF-κB that in turn pro-motes IL-1β excessive secretion ([11, 29, 54–56]. Additionally, IL-1β

Fig. 6. Effect of olopatadine on the corneal epithelial immunohistochemical localization of the NF-κB-p65 in male Wister rats' alkali-induced corneal injury. A,Negative control without primary antibody showed no immune reaction (→). B, CON group, showed epithelial cells with positive cytoplasmic NF-κB-p65 immunereaction (→). C, CI group, revealed epithelial cells with positive nuclear NF-κB-65 reaction rather than cytoplasmic reaction (→). D, VEH group, showed the nuclearNF-κB-p65 reaction of the epithelial cells rather than cytoplasmic reaction (→). E, OL group, showed nuclear (→) and cytoplasmic (►) immune reactions of NF-κB-p65 (→). F, OH group, showed epithelial cells with positive cytoplasmic NF-κB-65 immune reaction (→) (NF-κB-65. Scale bar= 50 μm).


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provokes apoptosis by activation of caspase-3, thus entering in a viciouscircle, with consequent augmentation of the inflammatory cascades,apoptosis and corneal opacification [57, 58]. In the present research,corneal apoptosis saw to be significantly increased by NaOH topicalapplication, as indicated by the significant elevation of corneal caspase-3 level, as well as, the deeply stained epithelial cell nuclei in H & Estained sections when compared with the control group, which was inparallel with the augmented corneal IL-1β level and NF-κB nuclearexpression. However, the daily topical application of olopatadine ef-fectively and dose-dependently reduced the elevated corneal caspase-3level induced by alkali injury. This can be explained on the basis ofolopatadine inhibition of IL-1β release and NF-κB activation, withsubsequent suppression of apoptosis induction.

NF-κB-p65 expression, in the present study, exposed nuclear ratherthan cytoplasmic immunohistochemical reaction, in CI group, whencompared to the control group. These findings are consistent with theother findings of our present research, which proved that prolonged andsustained NF-κB-p65 upregulation is associated with corneal angio-genesis, as well as, ocular inflammation and opacification. This couldbe explained by the fact that NF-κB is a heterodimer complex consistingof two subunits, p50 and p65, that exist in the cytoplasm in an inactiveform associated with an inhibitory subunit IκB-α. Stimulation of NF-κBby the inflammatory cytokines induces dissociation of NF-κB from itsIκB-α inhibitory subunit [59]. So, allows NF-κB to migrate from thecytoplasm to the nucleus, where it binds to the promoters of NF-κB-regulated genes, initiating gene transcription, with augmentation of IL-1β expression [55]. Other studies, demonstrated that IL-1β promotesexpression and activation of NF-κB-p65, thus entering into vicious circleof inflammatory reactions amplification, with consequent delaying ofcorneal wound healing, corneal opacification, neovascularization thatend eventually into loss of vision [38, 39, 43]. Additionally, the in-hibition of inflammatory cascades mediated by NF-kB endorses cornealalkali wound healing, through suppression of the augmented IL-1βexpression [60]. Also, the increased NF-κB-p65 levels proved to be as-sociated with allergic eye diseases such as allergic keratoconjunctivitiswhich is triggered by degranulation of mast cells [38].

Data of the present work showed that olopatadine, in a dose-de-pendent manner endorsed the suppression of the expression, activation,and translocation of nuclear NF-κB from the cytoplasm to the nucleus,thus, repressed IL-1β expression, hence, promoted cornel alkali injuryhealing, with the restoration of corneal transparency, and hinderingcorneal neovascularization. The downregulation of NF-kB by olopata-dine can be explained by the activation of IκBα inhibitory subunitwhich has an intrinsic nuclear localization signal, that enters to thenucleus, and then displaces NF-κB from its nuclear DNA binding sites,so transports NF-κB back to the cytoplasm [59]. By suppression of theNF-kB activation, the corneal epithelial proliferation encouragedcausing corneal wound healing, with the restoration of normal cornealavascular transparency. However, the significant elevation of caspase-3level and nuclear translocation of NF-κB-p65 by the end of the 14th dayin the OH group, as compared to the control group, could be elucidatedby that these parameters measured at its expression peak-time, wherethe healing process is at its summit, and before its repression [11, 22,37, 50, 51, 56].

In conclusion, the daily topical olopatadine therapy effectively anddose-dependently attenuated alkali-induced corneal injury through itsanti-inflammatory, anti-angiogenic, and anti-apoptotic activities, aswell as its suppressive ability on NF-kB-p65 expression and activation,which subsequently suppressed the corneal inflammation, opacity, andneovascularization, with abrogation of the histopathological changesinduced by alkali injury. Moreover, further investigations are in pro-gress to evaluate the corneal histopathological changes, the progressionof wound healing and rats' visual function throughout the course ofolopatadine therapy. So, olopatadine could be a potential medication totreat the alkali-induced corneal injury.

Funding

This research did not receive any grant from funding agencies in thepublic, commercial, or not-for-profit sectors.

Conflict of interest

The authors declare that they have no conflict of interest.

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Olopatadine enhances recovery of alkali-induced corneal