Current Eye Research, Early Online, 1–10, 2014! Informa Healthcare USA, Inc.

ISSN: 0271-3683 print / 1460-2202 online

DOI: 10.3109/02713683.2014.914540

ORIGINAL ARTICLE

Effect of Melatonin and Analogues on Corneal WoundHealing: Involvement of Mt2 Melatonin Receptor

Almudena Crooke1, Ana Guzman-Aranguez1, Aranzazu Mediero1,Pilar Alarma-Estrany1, Gonzalo Carracedo2, Teresa Pelaez1,

Assumpta Peral2 and Jesus Pintor1

1Departamento de Bioquımica y Biologıa Molecular IV, Facultad de Optica y Optometrıa,Universidad Complutense de Madrid, Madrid, Spain, and , 2Departamento de Optica II,Facultad de Optica y Optometrıa, Universidad Complutense de Madrid, Madrid, Spain

ABSTRACT

Purpose: We have investigated the effect of melatonin and its analogues on rabbit corneal epithelial woundhealing.

Methods: New Zealand rabbits were anaesthetised and wounds were made by placing Whatman paper discssoaked in n-heptanol on the cornea. Melatonin and analogues (all 10 nmol) were instilled. Wound diameterwas measured every 2 hours by means of fluorescein application with a Topcon SL-8Z slit lamp. Melatoninantagonists (all 10 nmol) were applied 2 hours before the application of the n-heptanol-soaked disc and thenevery 6 hours together with melatonin. To confirm the presence of MT2 receptors in corneal epithelial cellsimmunohistochemistry, Western blot and RT-PCR assays in native tissue and in rabbit corneal epithelialcells were performed. The tear components were extracted then processed by HPLC to quantify melatoninin tears.

Results: Migration assays revealed that melatonin and particularly the treatment with the MT2 agonist IIK7,accelerated the rate of healing (p50.001). The application of the non-selective melatonin receptor antagonistluzindole and the MT2 antagonist DH97 (but not prazosin), prevented the effect of melatonin on woundhealing (both p50.001). Immunohistochemistry, Western blot and RT-PCR assays showed the presence of MT2

melatonin receptor in corneal epithelial cells. In addition, we have identified melatonin in tears and determinedits daily variations.

Conclusions: These data suggest that MT2 receptors are implicated in the effect of melatonin on corneal woundhealing regulating migration rate. This suggests the potential use of melatonin and its analogues to enhanceepithelial wound healing in ocular surface disease.

Keywords: Corneal wound healing, melatonin, melatonin agonists, melatonin antagonists, melatonin receptors

INTRODUCTION

Melatonin is an indolamine produced by the pinealgland; it participates in many functions including thecoordination of circadian rhythms.1–5 Some functionsof melatonin are mediated by stimulating MT1 andMT2 receptors as well as the putative MT3 receptor6–8,while others may involve nuclear binding sites9,10 orbeing receptor independent.11,12 Although melatonin

is most widely known as a product of the pinealgland13, melatonin synthesis also occurs in extra-pineal sites such as the eye.14,15 Melatonin is synthe-sized by retinal photoreceptors and ciliary epithelialcells16,17 and it modulates a variety of physiologicalprocesses in the eye. Particularly, in retina, melatoninmodulates the photoreceptor outer segment sheddingrate18, horizontal cell sensitivity to light19, and dopa-mine release.20 In the anterior chamber, melatonin

Correspondence: Jesus Pintor, Departamento de Bioquımica y Biologıa Molecular IV, Facultad de Optica y Optometrıa, UniversidadComplutense de Madrid, C/Arcos de Jalon 118, 28037 Madrid, Spain. Tel: +34-91-3946859. Fax:+34-91-3946885. E-mail: jpintor@vet.ucm.es

Received 2 December 2013; revised 13 March 2014; accepted 02 April 2014; published online 14 May 2014

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regulates the aqueous humour balance determiningthe physiological parameter termed intraocular pres-sure (IOP).21,22

Less information is available about the effectsof melatonin on the ocular surface. The presence ofthis indole in tears remains unclear. MT1 melatoninreceptor expression has been detected in the cornealepithelium of Xenopus laevis, chick and human.23–25

In contrast, the presence of Mel1b (homologous to themammalian MT2 receptor) and Mel1c (homologous tothe mammalian GPR50, G protein-coupled receptor50) receptors only has been described in cornealepithelium of Xenopus laevis.26,27

Potential roles for melatonin in the cornea includethe modulation of corneal hydration state28, increasein tear secretion29 and protection against reactiveoxygen species30. Moreover, a role for melatonin incorneal growth and repair has been suggested byparallels between the retinal melatonin rhythmand the rhythms in corneal epithelial mitotic activ-ity.31 The renewal of the corneal epithelium has beenshown to exhibit a circadian regulation in the mitoticrate of corneal epithelial cells, which is high duringthe night and low during the day.32 This circadianvariation also affects the rate of corneal epithelialwound healing.33 On the other hand, it has beenreported that melatonin exerts a positive effect onwound repair after skin injury34 and dermal woundhealing was prolonged in experimental animalsdeprived of melatonin due to pinealectomy.35 Thesefindings hypothesize the beneficial action of mela-tonin in corneal wound healing.

To elucidate this issue, in this work, we describe theeffect of melatonin on corneal wound healing aftera corneal injury in New Zealand white rabbits.In addition, we assess the levels of melatonin in tears.

MATERIALS AND METHODS

Animals

Adult New Zealand white rabbits (males, 2–3 kg)were used. The animals were kept in individual cageswith free access to food and water, under controlledcycles (12 h light/12 h dark). Experiments werecarried out in accordance with the EuropeanCommunities Council Directive (86/609/EEC) andthe statement of the Association for Research inVision and Ophthalmology on the Use of Animals inOphthalmic and Vision Research.

Cell Culture

The rabbit corneal cell line, SIRC (StatensSeruminstitut Rabbit Cornea), was obtained fromATCC (LGC Promochem SL., Barcelona, Spain).

SIRC cells were maintained in minimum essentialmedium (MEM) with Earle’s salts, L-glutamine andnon-essential aminoacids (Invitrogen, Paisley, UK)supplemented with 10% activated Foetal BovineSerum (Invitrogen), and incubated at 37 �C in 5%CO2 and 95% humidity until confluence.

Corneal epithelial wound healing assay

Corneal wounds were made in both eyes by anaes-thetising the animals with propofol (1.5 mg/kg ofbody mass; Abbott Laboratories, Madrid, Spain).After topical anaesthesia with 0.4% oxybuprocaineand 1% tetracaine (Alcon Cusi, Barcelona, Spain),corneal wounds were made to the epithelia of botheyes by applying a 3 mM disc of Whatman no. 1 papersoaked in n-heptanol (Merck, Darmstadt, Germany).Discs were placed in the centre of the cornea and leftthere for 30 seconds. 36 Eyes were then washed threetimes with 5 ml of 0.9% NaCl solution. We applied10 ll of melatonin (10 nmol in 0.9% NaCl) to oneeye and 10 ll of 0.9% NaCl to the contralateral eye(control) of each animal (n = 8) every 6 hours between10 and 24 hours after wounding. Control animals(n = 8) were only treated in one eye with salinesolution, as previously described for rabbits treatedwith melatonin.

Antagonist effect of luzindole, DH97 and prazosin(all 10 nmol, 10 ll) on melatonin-mediated woundhealing effect was also assayed. These antagonistswere applied 2 hours before the application ofmelatonin and then every 6 hours together withmelatonin, as described above.

To evaluate the closure of the wounds, corneaswere stained with 2% fluorescein and examined witha Topcon SL-8Z slit lamp (Topcon Spain, Madrid,Spain) every 2 hours between 10 and 24 hours afterwounding. Images were taken, managed and ana-lysed with the IMAGEnet 2000 system (Topcon).

To model the decrease in wound area duringcorneal epithelial healing, the constant velocitymethod described by Crosson and colleagues wasused.37 Briefly, estimated migration rates (EMR) weredetermined by linear regression of the decrease inwound radius during the in vivo linear healing phase(10–24 hours after wounding) and were obtained bythe slope of the regression line expressed as microm-eters per hour. The total time of wound closure(estimated healing time, EHT) was calculated byextrapolation of the best fit of regression lines duringthe healing phase to 100% closure for each eye tested.

Immunohistochemistry (IHC) procedure

Immunofluorescent staining was performed to evalu-ate expression and location of MT2 melatonin

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receptors in corneal slices of healthy rabbits (n = 2).Animals eyes were fixed overnight at 4 �C, using asolution of paraformaldehyde 4% in 0.15 M phos-phate-buffered saline (PBS) (pH 7.2) and hemisectedhorizontally. Sections of 10 mM were made usinga Leica 3050 M cryostate (Leica microscopy system,Heerbrugg, Switzerland). Cornea sections wereembedded in OCT (Leica microscopy system,Heerbrugg, Switzerland) cryoprotective medium,washed with PBS 10X, and permeabilized withblocking solution (PBS 1X BSA 3% Triton X-100 FBS5%) for 1 hour at 37 �C. Sections were then washedwith PBS 1X BSA 3% and incubated with primary goatpolyclonal anti-MT2 (Santa Cruz Biotechnology Inc.,Santa Cruz, CA, USA) 1:100 or PBS 1X BSA 3% fornegative controls at room temperature for 1 hour.A wash was made twice in PBS 1X BSA 3% andincubation with the secondary antibody goat antimouse IgG-TRICT (Sigma) (1:200) for 1 hour at roomtemperature was performed. Three washes in PBS 1Xwere performed and coverslips were applied to theslides with mounting medium (Invitrogen). Sectionswere observed by confocal microscope (Axiovert200M; Carl Zeiss Meditec GmbH, Jena, Germany),equipped with a Pascal confocal module (LSM 5;Zeiss). All images were managed with the accom-panying Pascal software.

Western blot analyses

For western blot analysis, SIRC confluent monolayerswere collected in PBS. Cells were lysed and homo-genized in protein extraction buffer containing 20 mMTris pH 7.5, 150 mM NaCl, Triton X-100 (comprising1 mM PMSF, 1 mM NaF, 1 mM Na3VO4, 1 mg/mlPepstatin A, 2 mg/ml Leupeptin and 1 mg/mlAprotinin)(Sigma). After centrifugation (1549 Xg, 20minutes, 4 �C), the supernatants were collected andprotein concentration determined by Bradford proteinassay (Bio-Rad, Madrid, Spain). Forty-five micro-grams of protein were subjected to 10% SDS-PAGEelectrophoresis (Bio-Rad) and transferred to a nitro-cellulose membrane (Amersham Biosciences,Barcelona, Spain). To block the non-specific binding,the membranes were treated with PBS with 5%skimmed milk, and then they were incubated over-night with primary goat polyclonal anti-MT2 (SantaCruz) diluted 1:1000 in PBS – Tween 20 0.05%containing 2% skimmed milk. Membranes were thenwashed three times (10 minutes per wash) with PBS –Tween 20 0.05%, and further incubated with thedonkey antigoat IgG-HRP secondary antibodydiluted 1:2000 (Santa Cruz). Proteins were visualizedby ECL detection system (Amersham) and imagedusing a KODAK Gel Logic 2000 and KODAKMolecular Imaging Software (KODAK, Grupo Taper,Alcobendas, Madrid, Spain).

Reverse-transcriptase polymerase chainreaction (RT-PCR) analysis

To analyse the expression of MT2 melatonin receptormRNA in rabbit corneal epithelium, total RNA wasextracted from SIRC cells using the RNeasy Mini Kit(Qiagen, Barcelona, Spain). For first-strand cDNAsynthesis, 1.5 lg of total RNA was retrotranscribedusing High Capacity cDNA RT kit (AppliedBiosystems). The MT2 melatonin receptor (MT2-R)degenerate forward primer 50-CAACCTCCTGGTSATCCTCTC-30 and MT2-R specific reverse primer50-GACCACTACTGCCGCTGTGTA-30 were used inRT-PCR amplification. PCR amplification was per-formed in a 50 ll volume with 2 ll of cDNA, 1X PCRbuffer, 2 mM MgCl2, 200 lM each dNTPs, 0.4 lM ofeach primer, and 0.025 U/ll of AmpliTaq Gold� DNApolymerase (Applied Biosystems, Foster City, CA).The thermal cycling conditions for PCR were 95 �Cfor 8 min; 35 cycles of 95 �C for 30 s, 60 �C for 30 s,and 72 �C for 45 s each; and 1 cycle of 72 �C for 7 min.Non-template and non-reverse transcribed controlswere included in PCR reaction. The PCR productof the expected size was extracted from 1.5% low-meltagarose gels with the QIAquick Gel Extraction Kit(Qiagen), cloned with TOPO TA Cloning Kit(Invitrogen), and then sequenced. DNA sequencingwas performed by the Unidad de Genomica (ParqueCientıfico de Madrid-Universidad Complutense,Madrid, Spain). The nucleotide sequence was com-pared by searching the GeneBank databases with theBLAST program. Alignment of amino acid sequenceswere performed with the Clustal W2 program usingdefault parameters.38

Tear collection and chromatographicprocedures

The tear components were extracted from tears usingWhatman no. 51 paper strips (Schirmer strips;Whatman, Maidstone, UK) placed in the inferioreyelid margin of the awake animals for 5 minutes.Melatonin was measured at 4 times, once in themorning, just when the light was on (8:00 h), at12:00 h, in the evening just before the lights were off(20:00 h), and another in the middle of the night(2:00 h)(n = 6). Collection of tear samples at 20:00 h andat 2:00 h was performed under red light, to avoidlight-induced suppression of melatonin synthesis.2

Tear samples were then placed in Eppendorf tubescontaining 500ml of ultrapure water and stronglyvortexed for 5 minutes. The Schirmer strips werecarefully rinsed and the liquid in the tube wasprocessed according to the protocol described byKulczykowska and Iuvone.39

Exactly, 100 microliters of the sampleswere injected into the HPLC for analysis.

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The chromatographic system consisted of a WatersmBondapak C18 column (25 cm in length, 0.4 cm indiameter), a 1515 Isocratic HPLC pump, a 2487 dualabsorbance detector, and a Reodyne injector, allmanaged by the software Breeze from Waters(Milford, MA). The system was equilibrated overnightwith 60% methanol, 40% H2O. Measurements wereperformed at a flow rate of 0.6 ml/min fixing thedetector at a wavelength of 229 nm.39 Quantificationof melatonin was performed by comparing the sam-ples with external standards provided by Sigma.

Statistical analysis

Average values of in vivo corneal wound healingassays were expressed as the mean ± S.E.M. EMRand EHT determined in treated and control eyeswere compared using ANOVA test. Tear secretionstatistical analysis was performed using pairedStudent’s t-test. The levels of significance for thedifferences are indicated in each case in the figurelegends.

RESULTS

Corneal epithelial wound healing assays

The wound healing process in eyes of animals onlytreated with saline solution (control animals), wassimilar to that observed in contra lateral eyes (saline-treated eyes) of animals tested with both melatonin aswell as saline solution (results not shown). Therefore,a possible bilateral effect of unilaterally administeredmelatonin was ruled out and the process observed insaline-treated eye was considered control situation.

As observed in Figure 1, the wound healing processin control situation is linear and from the slope it ispossible to calculate the rate of re-epithelialization.Under this control situation, the rate of healing orEMR was 75 ± 5 mm/hour and the EHT was 29.8 ± 1.9hours (Table 1). When the same experiment wasperformed by applying single doses of melatoninupon the wounds (10 nmol in 10 ml), it was possible toobserve a clear increase in the EMR by 35 mm/hourwith respect to control situation (Figure 1B andTable 1). The associated EHT in eyes treated with

FIGURE 1 Effect of melatonin and its analogues on rabbit corneal wound healing. (A) Shows a representative sequence of the progressin the corneal wound closure of eyes treated with melatonin (10 nmol, 10 ll) or saline solution (control). The photographs wereobtained after 10, 12, 14, 18, 22 and 24 hours of wounding. (B) The graph shows the variation of the wounded area versus time aftertreatment with melatonin, IIK7 and 5-MCA-NAT (all 10 nmol, 10 ll) or saline solution (control). Both melatonin and IIK7 increasedEMR and reduced EHT when compared to control. Conversely, 5-MCA-NAT had not effect in comparison with control situation. Eachdata point represents mean ± S.E.M. of eight eyes.

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melatonin was reduced in 9.4 hours when wecompared with control eyes (Table 1).

The MT2 melatonin receptor agonist IIK7 presenteda similar action to that observed for melatonin(Figure 1B and Table 1). On the contrary, theMT3 melatonin receptor agonist 5-MCA-NAT, didnot modify the rate of healing as it can be seenin Table 1.

We have tested the effect of the non-selectivemelatonin receptor antagonist luzindole, the MT2

antagonist DH97 and the MT3 antagonist prazosinon rabbit corneal wound healing, in the presence ofmelatonin (all 10 nmol in 10 ml). Luzindole was able todelay the rate of healing induced by melatonin in58 mm/hour (Figure 2 and Table 1). Furthermore,an increase in the time of wound closure of near to17 hours was observed in eyes treated with luzindoleplus melatonin (Table 1). In addition, we appliedluzindole in the absence of melatonin and a reductionin the rate of re-epithelialization was also observed(result not shown).

The selective MT2 melatonin receptor antagonistDH97 prevented the wound healing effect of mela-tonin decreasing EMR (p50.001) and increasing theassociated time of wound closure in close to 20 hoursin comparison with animals treated with melatoninalone (Figure 2 and Table 1). On the contrary, andas we have shown in Figure 2 and Table 1 the MT3

melatonin receptor antagonist prazosin40, did notmodify the rate of healing in comparison with animalstreated with melatonin alone.

FIGURE 2 Effect of melatonin receptor antagonists on rabbit corneal wound healing. (A) Shows a representative sequence of theprogress in the corneal wound closure of eyes treated with the melatonin receptor antagonist luzindole, DH97 and prazosin plusmelatonin (all 10 nmol, 10 ll). The micrographs were obtained after 10, 12, 14, 18, 22 and 24 hours of wounding. (B) The graphs showthe variation of the wounded area versus time after treatment with melatonin alone, melatonin receptor antagonists plus melatonin(all 10 nmol, 10 ll) or saline solution (control). Both luzindole and DH97 reduced EMR and increased EHT when compared to eyestreated with melatonin alone. Each data point represents mean ± S.E.M. of eight eyes.

TABLE 1. EMR and EHT for melatonin receptor agonists andantagonists in rabbit corneal wound healing.

Compound EMR (mm/hour) D EHT (hours)

Control 75 ± 5 0Melatonin 110 ± 7### �9.4IIK7 107 ± 2### �9.15-MCA-NAT 70 ± 3 +1Luzindole + Melatonin 52 ± 4*** +7.5DH97 + Melatonin 40 ± 6*** + 10.8Prazosin + Melatonin 105 ± 5 �8.9

Data are the mean ± S.E.M. of eight independent experimentsfor each treatment. For DEHT, + is a delay and � is anacceleration over control situation. ###p50.001 versus control;***p50.001 versus melatonin alone.

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Presence of MT2 melatonin receptorin rabbit corneal epithelial cells

To confirm the presence of MT2 receptors in cornealepithelial cells which can mediate the acceleratingeffect of melatonin, we performed IHC, Western blotand RT-PCR assays in native tissue and immortalizedSIRC cells. As shown in Figure 3A, the MT2 receptoris present in the three corneal layers: epithelium,stroma and endothelium.

Furthermore, a single band of the expected molecu-lar weight was obtained for both, western blot(42 kDa) and RT-PCR (536 pb) assays (Figures 3Band C, respectively). DNA sequencing of the sup-posed MT2 RT-PCR product confirmed its identity asnucleotide sequence shared 84% homology withhuman MT2 melatonin receptor sequence (result notshown).

Presence on melatonin in rabbits tears

Luzindole alone evoked a reduction in the rate ofre-epithelialization which seems to suggest the pres-ence of melatonin in tears. To confirm this and toevaluate whether its levels follow a circadian pattern,

melatonin concentration in rabbit tears was measuredat four different moments along the day. As it ispresented in Figure 4, melatonin values (37.2 ± 3.9 nM)in the morning decreased as the day progresseswhile during the noon the value was 34.4 ± 7.3 nM.However, melatonin values were significantlyincreased when measured at night, this value being42.6 ± 2.8 nM at evening (20:00 h) and 49.8 ± 3.5 nMat night (2:00 h) (n = 6) (Figure 4B). Interestingly,when the analysis of the amount of melatonin andthe corresponding volume for any given day werestudied, it was possible to observe that melatoninlevels did not significantly change along the day-nightperiod (Figure 4C). Nevertheless, when studying thetear volume we could demonstrate that at 12 pmthe tear volume mean was 11.2 ± 1.2 lL, while at 2 amtear secretion was 6.9 ± 1.1 lL (n = 6).

DISCUSSION

The healing of corneal wound is essential for main-tenance of corneal structure and function. Injury of thecorneal epithelium induces migration of the remain-ing epithelial cells surrounding the wounds to coverthe wounded area. In the present experimental workwe demonstrated that topical application of melatonincan accelerate corneal wound healing in New Zealandrabbits, this effect being blocked by the use ofmelatonin receptor antagonists. When melatonin isapplied to corneal wounds the estimated migrationrate increased and concomitantly the estimated heal-ing time was reduced as compared to control situ-ation. Crosson and colleagues have previouslydescribed the biphasic kinetic of rabbit cornealwound healing process37. These authors demon-strated that the epithelial wound closure in therabbit cornea has a linear phase between 8–30 hoursafter wounding which slope is the rate of healingprocess. In our case, we have calculated the estimatedmigration rate in the period from 10 to 24 hours afterwounding. The variation of the wounded area versustime after treatment with melatonin can appearsmore logarithmic than linear due to the area observedbetween 14 and 20 hours. A possible explanation ofthis effect could be a different size of the initial woundof eyes treated with melatonin in comparison witheyes treated with saline solution. Nevertheless,Crosson and colleagues have proved that migrationrate is unaffected by variations in the initial woundsize.37 On the other hand, it could be the case thatendogenous melatonin released into the tear wouldimprove the healing action of exogenous melatonin.In fact Djeridane and colleagues have shown thatHarderian gland, an accessory lacrimal gland presentin some animals, synthetizes and releases melatonin41.

Several authors have used the Crosson’s methodto evaluate the healing effect of different substances

FIGURE 3 Presence of MT2 melatonin receptors in rabbitcorneal epithelial cells. (A) Confocal images of IHC of rabbitcorneal tissue section with anti-MT2 antibody. Nomarski imageof rabbit corneal sections is also shown. (B) Western blotanalysis of SIRC cells using anti-MT2 antibody. M, molecularweight marker (kDa); Lane 1, contains 45 lg of total proteinobtained from SIRC cells. Image shows a single band in themolecular weight range expected for MT2 receptor protein.(C) RT-PCR analysis of SIRC cells using MT2-R degenerateforward primer and MT2-R specific reverse primer. M, 100 bpDNA ladder molecular weight marker (pb); Lane 1, RT-PCRproduct obtained from total RNA (1.5 lg) of SIRC cells; Lane 2,RT-PCR product obtained from total RNA (1.5 lg) of SIRC cellsnon-reverse transcribed (negative control). RT-PCR amplifica-tion yielded a single band of the expected size.

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in rabbit cornea.42–44 In this context, melatonin hasan intermediate healing action between uracil- andadenosine- nucleotide derivatives (DEMR melatonin-

saline = 35 mm/hour; DEMR UTP-saline = 49.2 mm/hourand DEMR Ap4A-saline = 21.3mm/hour)43.

While this is the first time that the positive actionof melatonin on corneal wound healing has beenshown, previous studies have already analysed theeffect of melatonin on the wound healing of othertissues. Thus, administration of melatonin to rats withskin incisions significantly improved wound heal-ing.45 Similarly, melatonin accelerated the process ofwound repair in full-thickness incisional wounds ina rat model of dermal wound healing, improving thequality of scarring, both in terms of maturity andorientation of collagen fibres.34 In addition, melatoninwas also able to increase the rate of healing of colonicanastomosis in a rat model of peritonitis 46 as well asthe healing of idiopathic gastro-duodenal ulcerswith Helicobacter pylori infection in patients.47

The involvement of this neurohormone in woundhealing process has also been studied in pinealecto-mized rats, consequently, deprived of melatonin.In these pinealectomized rats, the rate of skinwound healing was clearly delayed.35,48 This delayin the rate of healing was reversed when thepinealectomized rats were treated with melatonin.48,49

Supporting this notion, melatonin administration topinealectomized rats also reversed the effect of pine-alectomy in infarcted heart scar.49

There are other studies indicating that melatonincan produce the opposite effect on wound healing.Melatonin caused inhibition of cell migration bychanging cytoskeletal organization via ROCK path-way in breast cancer MCF-7 cells.50 In addition, it hasbeen reported that exogenous melatonin exertednegative effects on wound healing by decreasedcollagen synthesis and epithelium proliferation inboth normal and pinealectomized rats with incisionwounds.51 Likewise, no significant effect on thehealing of colonic anastomoses was found in pine-alectomized rats.52 Factors such as the concentrationof melatonin used in the experiments or the time ofapplication could justify, in part, the discrepancyfound among several studies. Additionally, thesecontroversial results suggest that melatonin effectcan differ on the target organ depending on thereceptor which is activated.

In our experiments, the MT2 receptor seems to bethe responsible for mediating the effect of melatoninin re-epithelialisation according to the pharmaco-logical profile obtained using specific MT2 agonist(IIK7) and MT3 agonist (5-MCA-NAT) as well asspecific MT2 and MT3 antagonists (DH97 and

FIGURE 4 Presence of melatonin in rabbit tears. (A) HPLC profile of melatonin in rabbit tears collected at noon (12:00 h) and midnight(2:00 h). Samples analyzed as described under Materials and Methods section presented a putative peak identified as melatonin whencompared with commercial standard (scale for the samples 0.001 AUFS). (B) The graph shows the behavior of melatonin concentrationin rabbit tears with a maximal peak of melatonin at night. (C) The amount of melatonin released to the tear at different moments of theday and night, showing the minimal changes in the amount of the released neurohormone and indicating that the changes in B aremostly due to variations in the tear volume. Each data point represents mean ± S.E.M. of eight eyes. *p50.05 compared withcorresponding noon value.

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prazosin, respectively). Moreover, we confirmed, forthe first time, the presence of the MT2 receptor inrabbit corneal epithelium using different techniques(IHC, western blot and RT-PCR).

It is important to note that we cannot discard thepossible participation of other melatonin receptorssuch as the MT1. In fact, IIk7 is a full agonist at theMT2 receptor but a partial agonist at the MT1 receptor.Therefore, the effects of IIK7 on epithelial migrationmay be in part due to its interaction with MT1

receptor. Nevertheless, it is necessary to keep in mindthat its affinity for MT2 receptor is 90-fold higherthan for MT1 receptor.53 In addition, the applicationof the melatonin receptor antagonist luzindole whichhas approximately 11- to 25-fold greater affinity forthe MT2 over the MT1 receptor 54,55 prevented mela-tonin-induced corneal wound healing. Nonetheless,the specific MT2 antagonist DH97 56 prevented thismelatonin effect in a greater extent than luzindole.

Interestingly, we observed that the treatment withthe antagonist luzindole, in the absence of melatoninapplication, delayed the rate of re-epithelialisation.This fact matches well with the presence of melatoninin tears along the day, that we demonstrated byHPLC. Until now four anatomical locations havebeen proved to synthesize melatonin: pineal gland,retina, ciliary processes and Harderian gland (seecommentary above),16,17,41 but the exact source ofthis neurohormone in tears remains unclear. In fact,the possible existence of melatonin inside cornealepithelial cells cannot be ruled out since as we havedemonstrated, melatonin MT2 receptors are present inthese cells. Independently from the origin of rabbittear melatonin, it looks clear that it shows dailyvariations. The concentration during the night is40–45% higher than in the daytime, but surprisinglythe changes in the melatonin concentration were dueto the variations in the tear volume rather than inthe melatonin amount. Independent of the reasonthat produced the rise in melatonin concentrationduring the night, it matches with the variations of thisneurohormone as occurs in other tissues. In this sense,it has been reported that melatonin concentration inother bodily fluids, tissues, cells and even particularsubcellular organelles are several orders of magnitudegreater than those in the blood traditionally con-sidered as physiological levels.57–62 Thus, it is tempt-ing to speculate that this melatonin present in tearscould modulate biochemical processes importantfor the ocular surface, such as corneal mitoticrate or corneal re-epithelialisation. In this sense,a regular diurnal rhythm of light and dark periodsis essential for normal corneal development, andlocal mechanism appear to play a major role on theconstant-mediated effects on this growth.63

The renewal of corneal epithelium has been shownto exhibit a circadian regulation32 and these circadianvariations have been shown to affect corneal epithelial

wound healing.64 The mitotic rate of corneal epithelialcells exhibits high levels during the night and lowlevels during the day, with circadian variations inepithelial proliferation appearing to be most prevalentin peripheral regions in rat corneal epithelium.33

Thus, melatonin in tears may be responsible formediating circadian rhythms in corneal growth viainteraction with specific melatonin receptor subtypeson corneal epithelial.

In summary, we can conclude that melatonin viathe activation of the MT2 receptor is able to acceleratethe migration rate of wounded corneal epithelialcells suggesting the potential use of this compoundfor the treatment of corneal injuries.

DECLARATION OF INTEREST

The authors report no conflicts of interest. The authorsalone are responsible for the content and writing ofthe paper. This work was supported by grants fromthe Spanish Ministry of Economy and Competition[SAF2010-16024]; the Ministry of Health SocialServices and Equality RETICS [RD12/0034/0003];and the Universidad Complutense de Madrid[GR35/10-A-920777].

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