Current Eye Research, Early Online, 1–6, 2014! Informa Healthcare USA, Inc.

ISSN: 0271-3683 print / 1460-2202 online

DOI: 10.3109/02713683.2013.853195

SHORT COMMUNICATION

Acute Exposure of Rabbit Eyes to Artificial Light in Vivo:Effect on Corneal and Third Eyelid Conjunctival

Histology and the Gene Expression of PAFR

Efterpi Varsamidou1, Soultana Markopoulou1, Georgia Karayannopoulou2, AntonisKalpatsanidis3, Vassileios Kokkas1, Vassileios Karampatakis3 and Antonis Goulas1

1Department of Pharmacology, 2Department of Anatomical Pathology, and 3Laboratory of ExperimentalOphthalmology, Medical School, Aristotle University of Thessaloniki, Thessaloniki, Greece

ABSTRACT

Purpose: To study the effect of acute exposure of rabbit eyes to artificial sunlight in vivo, on the integrity ofcorneal and conjunctival tissue as well as on the gene expression of the receptor for platelet activating factor(PAFR).

Methods: New Zealand albino rabbits were immobilized opposite a 300 W Osram Ultra-Vitalux� light bulb withan emission radiation spectrum similar to that of normal sunlight at noon, and exposed to ultraviolet Bradiation in the range of the reported threshold for corneal damage. Corneal and third eyelid tissue sampleswere removed from exposed eyes at 2, 6 and 24 h following the end of the exposure to the bulb light and weresubsequently processed for histochemical staining and RNA extraction. The gene expression of PAFR wasdetected with real time reverse transcription polymerase chain reaction.

Results: Some epithelial shedding was detected in the corneal tissue as a result of acute exposure to artificialsunlight. In the eyelid conjunctiva, a marked accumulation of eosinophils was noticed, as early as 2 h post-exposure, apparently directed toward the upper part of the epithelial layer. This effect appears to subside byhour 24. No statistically significant changes in gene expression were detected in the corneal tissue, whereas inthe third eyelid, PAFR gene expression was significantly induced, most prominently at t = 2 and 6 h post-exposure.

Conclusion: Acute exposure of rabbit eyes to artificial sunlight induced a marked infiltration of eosinophils intothe epithelial layer of the conjunctiva but no gross alterations in the cornea or the third eyelid. The geneexpression of PAFR was upregulated, as an effect of light exposure, in the third eyelid but not in the cornea.

Keywords: Artificial sunlight, conjunctiva, cornea, eosinophils, gene expression, rabbit

INTRODUCTION

Acute exposure to solar or artificial radiation caninduce inflammatory changes in the anterior chamberof the eye and the conjunctival tissue.1,2 These changesmanifest themselves as photokeratoconjunctivitis, pro-ducing a variety of symptoms, which include itchiness,tearing, pain and photophobia, depending on theextent of the damage produced.3,4 The symptomsusually subside, one to two days following exposure,

but not before they cause enough discomfort to lead theaffected individual in search of medical treatment.5

Proper understanding of the mechanisms underlyingphotokeratoconjunctivitis at the tissue, cellular and themolecular level can help design efficacious pharmaco-logical intervention. Exposure thresholds for tissuedamage are wavelength-specific and at their lowestbelow 310 nm, within the ultraviolet B (UVB) range.1,6

At supra-threshold values, epithelial damage of thecornea can reportedly be detected as early as 2 h

Correspondence: Antonis Goulas, Department of Pharmacology, Medical School, Aristotle University of Thssaloniki, Thssaloniki, Greece.Tel/Fax: +302310 999342. E-mail: goulas@med.auth.gr

Received 18 January 2013; revised 18 September 2013; accepted 30 September 2013; published online 8 January 2014

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following exposure.6 UVB irradiation was shown in thepast to induce the gene expression of various cytokinesin human corneal and pterygial cells.7–9 UVB wasshown to generate platelet activating factor (PAF) andPAF analogs in an epidermal cell line.10,11 PAF exerts itseffects by interacting with a seven transmembrane-spanning G-coupled receptor (PAF receptor – PAFR),which is expressed in most ocular tissues12,13 and itsgene expression is increased as part of the response tocorneal injury.14

In this study, we investigated the early effects ofin vivo acute exposure of rabbit eyes to a source ofartificial light with an emission spectrum similar tothat of solar radiation by examining tissue structuralchanges as well as the gene expression of PAFR inthe cornea and the third eyelid of the New Zealandalbino rabbit.

MATERIALS AND METHODS

A total of 16 animals were used in this study. Eachrabbit was four months old and weighed approxi-mately 3.5 kg at the time of the experiments. Animalswere caged individually, under conditions of normalphotoperiod and temperature. Eating and drinkingwas ad libitum. Acute exposure to artificial light wasaccomplished by immobilizing each animal directlyopposite – at a distance of 60 cm – a 300 W OsramUltra-Vitalux� light bulb. The bulb, which is com-mercially available and commonly used for tanningpurposes, has a radiation emission spectrum similarto that of normal sunlight and an energy outputcomparable to solar recordings at noon, duringsummer in the northern hemisphere (manufacturer’sspecifications: http://www.svetila.com/eProdaja/product_info.php/products_id/1170). Total exposurein the UVB range (280–315 nm) for 30 min wasmeasured, by using a Brewer type MKIII monochrom-ator, at 0.75 J cm�2. A total of 12 animals were exposedto artificial light, while four were used as controls. Att = 2, 6 and 24 h following exposure, the animals wereanaesthetized and tissue samples of the cornea andthe nictitating membrane (third eyelid) were surgi-cally removed (n = 4 per each post-exposure timepoint, as well as for the non-irradiated control group)and either immediately frozen in liquid nitrogen andstored at �70 �C or placed into buffered 4% v/vformaldehyde solution and stored at room tempera-ture awaiting further processing for RNA isolation orhistopathological evaluation, respectively. Oculartissue was excised from either eye of each animaland processed individually. Corneas, in particular,were almost entirely removed, leaving only a narrowstrip by the corneoscleral border, and then cut intosmaller pieces with a scalpel. Overall, RNA wasisolated and successfully processed to produce cDNAfrom both eyes of three animals form each group (time

point/control), while ocular tissue samples from twoanimals per time point/group were histologicallyexamined. Only central, 0.3� 0.1 cm corneal frag-ments were used for histological examination. Allanimals were euthanized immediately following cor-neal removal. Animal handling was according to theguidelines of the European Communities CouncilDirective of 24/11/86 (86/609/EEC). The experimen-tal protocol was approved by the Aristotle UniversityMedical School Bioethics Committee.

For histopathological evaluation, tissue maintainedin formaldehyde solution was embedded in paraffin,sectioned at 5 mm and subsequently stained withhaematoxylin and eosin for light microscope examin-ation. Three histological sections have been assessedper sample block.

Total RNA was isolated from frozen corneas with aphenol-based reagent (TRIsureTM, BIOLINE, London,UK) according to the manufacturer’s specifications.Isolated RNA was rendered DNA-free through treat-ment with RNase-free DNase (TURBO DNA-freeTM

Kit; Invitrogen, Life Technologies Corporation,Carlsbad, CA) and quantified with a NanoDropTM

spectrophotometer (Thermo Scientific Inc., Waltham,MA). The RNA was then reversed transcribed intocDNA with SuperScriptTM II Reverse Transcriptase(Invitrogen, Life Technologies Corporation). polymer-ase chain reaction amplification of rabbit PAFRand glyceraldehyde-3-phosphate dehydrogenase(GAPDH) cDNA was accomplished using a TaqMan�

Universal Master Mix (Applied Biosystems, LifeTechnologies Corporation, Carlsbad, CA) on aStepOnePlusTM System (Applied Biosystems, LifeTechnologies Corporation) under the same thermalprofile: 95 �C for 20 s, followed by 45 cycles consistingof 95 �C for 1 s and 60 �C for 20 s. Primers and probe forPAFR amplification were designed using the PrimerExpress 3.0 program: forward, ACCAGGCTATTAATGATGCACACC; reverse, GTGCTTGCGGAACTTCTTGG; probe, TCCTTAGCACCAACTGT. For GAPDH, a TaqMan� Gene Expression AssayMix (Oc03823402_g1) was used (Applied Biosystems,Life Technologies Corporation). The 2�DDCt methodwas used for relative quantitation of PAFR geneexpression at the different time points.

The Mann–Whitney non-parametric test was used tocompare the relative gene expression of PAFR betweenthe control group of corneas and third eyelids and thoseexcised from the irradiated animals. p = 0.05 was set asthe limit for statistical significance. All statisticalanalyses were performed with the SPSS statisticalpackage (version 20; IBM, Armonk, NY, USA).

RESULTS

There were no macroscopic alterations worth notingin the eyes of the irradiated animals, except for some

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conjunctival hyperemia (not shown). Histopathologi-cal examination revealed no major changes inthe corneal architecture as a result of exposure toartificial light under the conditions employed in thisstudy (Figure 1A and B), with the exception ofsome epithelial shedding observed at t = 6 and 24 hpost-exposure (Figure 1C and D), and a small numberof cells with pycnotic nuclei and eosinophilic cyto-plasm detected in the corneal epithelium at t = 24 hpost-exposure (Figure 1D). On the other hand, adistinctive eosinophilic infiltration was observed – ascompared to untreated animals – as early as 2 hpost-exposure, in the conjunctiva of the third eye-lid, both in the epithelial layer and the basal lamina.In addition, a mild lymphocytic/plasmatocyticinfiltration was detectable in the basal lamina(Figure 2A–C). A marked depletion of goblet cellswas also observed by t = 6 h (Figure 2C). Cellularinfiltration had apparently subsided by t = 24 h(Figure 2D).

PAFR gene expression was detected in all tissuesamples, control and irradiated. A statistically signifi-cant difference with respect to the unexposed controlswas detected with respect to PAFR gene expression

in the third eyelid, which was more prominent att = 2 and 6 h following acute exposure to the bulblight (p = 0.002 and p = 0.004, respectively; Figure 3).An apparent transient increase of PAFR gene expres-sion in corneal tissue was not statistically significant(p = 0.394, t = 2 h; Figure 3).

DISCUSSION

Use of acute animal exposure experiments is thecurrently established method of studying light-induced ocular damage in vivo. In our study, weused a bulb with an emission spectrum similar to thatof sunlight to at least partially simulate conditions ofacute outdoors exposure. Experimental conditionswere selected as to produce a total exposure at the280–315 nm range, which is at the same order ofmagnitude as the corresponding threshold values forphotokeratitis reported by earlier investigators.1,15

The lack of extensive histological changes detectedin the cornea could be attributed to the fact that,at 0.75 J cm�2, this threshold was not effectivelyexceeded in our experiments even though the small

(A) (B)

(C) (D)

FIGURE 1. Eosin and hematoxylin staining of rabbit corneas. (A) cornea removed from control, non-irradiated animal;(B) cornea removed from animal 2 h post-exposure; (C) cornea removed from animal 6 h post-exposure; (D) cornea removedfrom animal 24 h post-exposure. Full-head arrows: epithelial shedding; pointed arrow: cells with eosinophil cytoplasm and pycnoticnuclei.

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number of epithelial cells with eosinophilic cytoplasmand pycnotic nuclei as well as the limited epithelialshedding attest to some damage related to UVexposure.16 On the other hand, it is at least equallyprobable that the nictitating membrane served toprotect the underlying cornea from more extensivedamage, as the same degree of exposure was able toinduce a distinctive cellular infiltration in the thirdeyelid conjunctiva, concomitant with a statisticallysignificant increase of PAFR gene expression.

The detection and the apparently directional natureof the eosinophilic infiltration into the conjunctivalepithelium merit some attention. While the presenceand possible role of eosinophils is generally notmentioned in the literature in connection to light-induced ocular damage, these inflammatory cells arereadily detected in at least one condition associatedwith chronic exposure to sunlight, namely actinicconjunctivitis. The latter constitutes part of actinicprurigo, a photosensitive reaction to UV light, whichinvolves – besides the conjunctiva – the skin and theoral mucosa, and that it apparently carries a geneticcomponent, as it almost exclusively affects people ofIndian ancestry in the Americas.17 Actinic conjunctiv-itis is characterized by additional tissue damage such

as epidermoid metaplasia, parakeratosis, loss ofgoblet cells and solar elastosis, but these changesneed time to manifest themselves and could notpossibly occur in a model of acute light exposure.Non-allergic eosinophilic conjunctivitis is anothercondition that involves limited eosinophilic infiltra-tion in the subepithelial layer of the conjunctiva andthat could be the result of chronic light exposure onsusceptible individuals.18

Understanding the mechanism by which light caninduce eosinophilic infiltration in the conjunctivalepithelium should allow better treatment of photo-conjunctivitis and of possibly related chronic condi-tions such as the ones mentioned above. Indeed, it hasbeen shown in the past that UVB irradiation is able toinduce the release of PAF from an epidermal cell line10

and of histamine from rat peritoneal mast cells19. Bothof these common proinflammatory mediators areknown to have chemoattracting properties towardeosinophils, histamine through its interaction withthe H4R receptor20 and PAF through binding to thePAFR.13,21 While the increase of PAFR expression inthe third eyelid appears to support the involvement ofPAF, future experiments should further examine thesepossibilities.

(A) (B)

(C) (D)

FIGURE 2. Eosin and hematoxylin staining of rabbit conjunctivas from the third eyelid. (A) third eyelid removed from control,non-irradiated animal; (B) third eyelid removed from animal 2 h post-exposure; (C) third eyelid removed from animal 6 h post-exposure; (D) third eyelid removed from animal 24 h post-exposure. Full-head arrows: eosinophils in the epithelial layer of theconjunctiva.

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DECLARATION OF INTEREST

The authors report no conflicts of interest. The authorsalone are responsible for the content and writing ofthis article.

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FIGURE 3. Relative expression of PAFR, normalized to that of GAPDH, in the corneas and third eyelids of non-irradiatedcontrol (Ctrl) and irradiated rabbits at t = 2, 6 and 24 h post-exposure (n = 3 pairs of eyes in each group/time point). Hatchedbars: corneas; solid black bars: third eyelids. Whiskers:� 95% confidence intervals. *: statistically significant difference with respect tocontrol.

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