We investigated the influence of alpha-melanocyte stimulating hormone (a-MSH) on the healing of corneal epithelial lesions in male Wistar rats. The lesions were induced by abrading corneal epithelium with a surgical lancet under a biomicroscope. Before the abrasion corneal epithelium was softened with 40% ethanol. a-MSH drops in a concentration of 1×10–4 mg/mL significantly promoted epithelial healing, when compared to the control group treated with 0.9% NaCl. Significant reduction of ocular irritation was also observed. Topical a-MSH medication could promote the healing of corneal wounds and reduce local inflammatory response without the major side effects of the corticosteroid therapy

Adrian Lukenda

Josip Pavan

Marko Curkovic

Nikola Stambuk

Pasko Konjevoda

Snjezana Kastelan

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Coll. Antropol. 36 (2012) 4: 1407–1411Original scientific paperEffects of Alpha-MSH on Corneal EpithelialLesions in RatsJosip Pavan1, Adrian Lukenda1, Nikola [tambuk2, Pa{ko Konjevoda2, Snje`ana Ka{telan1and Marko ]urkovi}11 University of Zagreb, Dubrava University Hospital, Department of Ophthalmology, Zagreb, Croatia.2 »Ru|er Bo{kovi}« Institute, Zagreb, CroatiaA B S T R A C TWe investigated the influence of alpha-melanocyte stimulating hormone (a-MSH) on the healing of corneal epitheliallesions in male Wistar rats. The lesions were induced by abrading corneal epithelium with a surgical lancet under abiomicroscope. Before the abrasion corneal epithelium was softened with 40% ethanol. a-MSH drops in a concentrationof 1×10–4 mg/mL significantly promoted epithelial healing, when compared to the control group treated with 0.9% NaCl.Significant reduction of ocular irritation was also observed. Topical a-MSH medication could promote the healing ofcorneal wounds and reduce local inflammatory response without the major side effects of the corticosteroid therapy.Key words: alpha-MSH, cornea, drops, epithelization, wound, healing, irritationIntroductionAlpha-melanocyte stimulating hormone (a-MSH) is apeptide deriving from the post-translational processingof the pro-opiomelanocortin (POMC) molecule1,2. ThePOMC prohormone gives rise to a variety of biologicallyactive peptides including adrenocorticotropin (ACTH)and a-MSH1,2. a-MSH corresponds to the first 13 aminoacids of the ACTH sequence. A synthetic peptide frag-ment of ACTH4–10 has been shown to improve the rabbitcornea wound healing3.Other results indicated that in addition to stronganti-inflammatory and cytoprotective effects on differentorgans and tissues a-MSH elicits antimicrobial and anti-fungal effects1,2,4,5.Impaired and prolonged reepithelization of corneal le-sions may result in long lasting corneal defects, whichcan possibly be followed by serious complications such asmicrobial and fungal inflammation and corneal opaci-fication. Therefore, we have investigated the effects ofa-MSH on the corneal healing and reduction of eye irri-tation caused by the abrasion of rat corneal epithelium.Materials and MethodsAnimalsWistar male albino rats weighing from 160 to 180 gwere used as the experimental animals. They were keptunder standard conditions with a 12 hour light/dark cy-cle. The rats were divided in groups of 6 animals and acontrol group had 8 animals.Corneal abrasionStandardized 3 mm wide circular lesions were in-duced by abrading corneal epithelium with a surgicallancet under a hand-held biomicroscope (Heine HSL100)6. Before scraping the cornea, epithelium was soft-ened with a circular filter paper soaked in 40% ethanol,placed on cornea for 30 seconds and irrigated with salinesolution. Abrasion procedure was done in light ether an-esthesia (Narkosaether, Lek, Slovenia). Immediately af-ter the abrasion, corneas were dyed with a sterile fluores-cein solution. High resolution digital camera (PanasonicNV-GX7, 1200000 pixels) was used to make the photo-graphs of abraded corneas under a cobalt blue UV lampat 0, 2, 8, 16 and 24 hour intervals.Treatment protocolCorneal lesions were treated with two eye drops ofa-MSH (SYSMEHFRWGKPV, >97% purity7 administer-ed in 10 seconds intervals, at 0, 2, 8 and 16 hours afterthe injury. The following eye drop concentrations wereused: 1 mg/mL, 1×10–1 mg/mL, 1×10–2 mg/mL, 1×10–31407Received for publication May 20, 2010U:\coll-antropolo\coll-antro-4-2012\10122 Pavan.vp21. prosinac 2012 11:31:04Color profile: DisabledComposite  150 lpi at 45 degreesmg/mL, 1×10–4 mg/mL. The control group was treatedwith 0.9% NaCl (1 drop = 0.05 mL).Evaluation and scoring of corneal lesionsand ocular irritationThe healing of corneal lesions was scored after the ini-tial abrasion using a modified Draize test6,8. Originally,Draize test is used to score the advancement of a lesionafter application of an irritant. In this experiment wehave reversed the method. The score was calculated asfollows: score 0 for no corneal lesion detected, score 1 for0–25%, score 2 for 25–50%, score 3 for 50–75% and score4 for 75–100% of initial lesion surface still not healed.The relative size of each lesion was compared to thewhole surface of the cornea using image processing soft-ware (ImageJ). The intensity of conjunctival irritationwas described by numbers, from 1 (for a minimal) to 10(for a maximal irritation as seen in saline treated eyes).The mean of the left and right eye score was used.Data analysisStatistical analysis and comparison of control andtreated groups was performed with Steel’s test – a non-parametric version of Dunett’s test. The analysis wasmade by using KyPlot software (version 4.0) and Graph-Pad Prism software (version 5.0).ResultsFive groups of experimental animals were treatedwith a-MSH drops in the following range of concentra-tions: 1 mg/mL, 1×10–1 mg/mL, 1×10–2 mg/mL, 1×10–3mg/mL and 1×10–4 mg/mL. The control group was treat-ed with 0.9% NaCl drops.a-MSH concentrations in the range from 1 mg/1 mLto 1?10–3 mg/mL did not significantly affect the rate ofcorneal healing (Table 1). The peptide concentration of1×10–4 mg/mL significantly promoted epithelial healingin rat corneas when compared to the control group, 16J. Pavan et al.: Alpha-MSH and Corneal Epithelial Lesions in Rats, Coll. Antropol. 36 (2012) 4: 1407–14111408TABLE 1EFFECTS OF DIFFERENT CONCENTRATIONS OF a-MSH ON THE HEALING OF CORNEAL EPITHELIAL DEFECTS 2, 8, 16 AND 24HOURS AFTER ABRASION (DESCRIPTIVE STATISTICS)Control (0.9% NaCl) N X Median Min Max Q1 Q3 SD Steel test2 h 8 99.8 101.4 60.6 140.6 87.1 109.9 24.28 h 8 68.9 70.1 22.3 92.6 65.0 83.0 21.516 h 8 43.7 38.2 16.8 84.0 29.3 56.9 22.624 h 8 16.3 14.0 2.7 49.0 7.6 17.9 14.2Alpha-MSH (1×10–4 mg/mL) N X Median Min Max Q1 Q3 SD p value2 h 6 75.7 72.5 53.4 110.5 70.6 74.8 18.8 0.6088 h 6 62.1 57.4 52.1 89.6 52.4 63.5 14.4 >0.99916 h 6 10.4 5.8 3.5 25.2 4.5 17.45 8.9 0.00824 h 6 0.1 0.0 0.0 0.6 0.3 0.6 0.2 0.007Alpha-MSH (1×10–3 mg/mL) N X Median Min Max Q1 Q3 SD p value2 h 6 78.5 78.8 58.8 99.6 68.1 86.7 14.2 >0.9998 h 6 66.9 70.9 42.7 85.3 56.4 75.3 15.3 >0.99916 h 6 24.9 23.5 7.6 42.1 10.1 38.5 14.2 >0.99924 h 6 1.2 0.0 0.0 5.2 0.00 3.7 2.1 0.001Alpha-MSH (1×10–2 mg/mL) N X Median Min Max Q1 Q3 SD p value2 h 6 85.4 85.8 69.4 103.5 76.4 91.4 12.1 >0.9998 h 6 57.0 58.2 43.3 68.4 51.9 62.2 8.7 >0.99916 h 6 20.2 19.8 8.2 36.8 8.5 27.9 11.1 0.69524 h 6 13.4 10.7 3.9 31.1 5.1 24.5 9.9 0.996Alpha-MSH (1×10–1 mg/mL) N X Median Min Max Q1 Q3 SD p value2 h 6 88.3 93.0 65.7 102.9 72.3 102.8 16.3 >0.9998 h 6 73.3 77.2 45.0 92.0 61.2 87.1 17.5 >0.99916 h 6 31.0 29.6 10.6 50.8 18.3 47.3 16.6 >0.99924 h 6 19.5 19.4 0.0 42.0 4.5 34.6 14.6 0.996Alpha-MSH (1mg/mL) N X Median Min Max Q1 Q3 SD p values2 h 6 9.0 113.8 73.4 138.1 95.3 119.7 22.2 >0.9998 h 6 75.7 77.9 50.6 91.1 74.5 82.1 13.7 >0.99916 h 6 24.6 24.3 13.9 41.3 14.9 28.8 10.2 >0.99924 h 6 17.3 18.7 5.2 25.6 7.8 25.4 8.2 0.853Alpha-MSH – alpha-melanocyte stimulating hormoneU:\coll-antropolo\coll-antro-4-2012\10122 Pavan.vp21. prosinac 2012 11:31:04Color profile: DisabledComposite  150 lpi at 45 degreesand 24 hours after epithelial abrasion (Table 1, Figure 1).a-MSH concentration of 1×10–3 mg/mL was also effec-tive 24 hours after epithelial abrasion (Table 1, Figure 1).Compared to the animals treated with 0.9% NaCl dropsa-MSH drops elicited a significant reduction of conjunc-tival hyperemia in all treated groups 16 hours after cor-neal abrasion (p<0.05, Table 2, Figure 2).DiscussionIntegrity of ocular surface epithelium and cornealre-epithelization is vital for normal hydration and trans-parency of the cornea. It may therefore have influence onvisual function and present a key process in preventionof corneal and intraocular infection9. During the healingprocess the migration and proliferation of the corneal ep-ithelium takes place. Simultaneously, activation of differ-ent growth factors as well as some cytokines occurs aspart of an inflammatory reaction10. Inflammatory andimmunological reactions inside the cornea may cause fi-brosis which could subsequently reduce corneal trans-parency and lead to blindness. Corticosteroids applied lo-cally have a beneficial effect on fibrosis prevention afterinjury. However, they also inhibit wound healing andthus can not be used until the integrity of the ocular sur-face is re-established. Moreover, they have a number ofknown side effects such as ocular hypertension, glau-coma, cataract formation, epithelial toxicity, crystallinekeratopathy, decreased wound strength and thereforethey should be applied very cautiously11,12.Although several substances have been reported toimprove the corneal healing rate6,12, it is still necessaryto search for substances that could facilitate cornealre-epithelization and prevent fibrosis at the same time.Besides the observed beneficial effects on corneal epithe-lization and irritation, a-MSH is known to play an im-portant role as a mediator of immunoregulatory andimmunosuppressory processes which occur in the aque-ous humor13. In anti-inflammatory reactions a-MSH isinvolved by suppression of prostaglandin E2 (PGE2), tu-mor necrosis factor-a (TNF-a), interleukin-1 (IL-1), in-terleukin-6 (IL-6) and monocyte chemotactic protein-1(MCP-1) production as well as by blocking cyclo-oxy-genase-2 (COX-2) expression14. Furthermore, it may alsobe useful as an anti-inflammatory agent in ocular trau-ma treatment maintaining blood-aqueous barrier integ-rity as opposed to steroids14. After penetrating corneo-limbal cut systemic and topical treatment with a-MSH inrabbits equal or better results in reducing intraocular in-flammatory reactions compared to topical application ofcorticosteroids were shown15. In the same animal modelits hypotensive ocular effect with sustained reduction ofintraocular pressure after a single topical applicationwas also established16. Moreover, the local subconjunc-tival treatment with a-MSH reduced allorejection of ratcorneal grafts17.J. Pavan et al.: Alpha-MSH and Corneal Epithelial Lesions in Rats, Coll. Antropol. 36 (2012) 4: 1407–14111409Control (0.9% NaCl)mg/mL)-4-MSH (1×10αmg/mL)-3-MSH (1×10αmg/mL)-2-MSH(1×10αmg/mL)-1-MSH(1×10αmg/mL)-MSH(1α0.02.55.07.510.012.515.017.520.022.525.027.5*** p < 0.05 vs. controlCornealepithelialdefectarea(%ofinitiallesion)Fig. 1. Healing of corneal defects – percentage of theinitial lesion after 24 hours. a-MSH – alpha-melanocytestimulating hormone.TABLE 2EFFECTS OF a-MSH ON OCULAR IRRITATION 16 HOURS AFTEREPITHELIAL ABRASION (INTENSITY SCORE FROM 1 FORMINIMAL TO 10 FOR MAXIMAL CONJUNCTIVAL HYPEREMIA)Substance X SD MedianSteeltest (p)Control 0.9% NaCl 9.7 1.22 10.0a-MSH 1 mg/mL 4.7 1.50 4.0 0.007a-MSH 1×10–4 mg/mL 5.2 1.47 5.5 0.007a-MSH 1×10–3 mg/mL 3.5 0.55 3.5 0.006a-MSH 1×10–2 mg/mL 5.2 0.98 5.5 0.007a-MSH 1×10–1 mg/mL 5.0 1.27 4.5 0.007a-MSH – alpha-melanocyte stimulating hormoneControl (0.9%NaCl)mg/mL)-4-MSH (1×10αmg/mL)-3-MSH (1×10αmg/mL)-2-MSH (1×10αmg/mL)-1-MSH (1×10α-MSH (1 mg/mL)α012345678910**** ** p < 0.05 vs. controlEyeirritation(0-10)Fig. 2. Ocular irritation in a-MSH treated groups and con-trols 16 hours after epithelial abrasion. a-MSH – alpha-melano-cyte stimulating hormone.U:\coll-antropolo\coll-antro-4-2012\10122 Pavan.vp21. prosinac 2012 11:31:04Color profile: DisabledComposite  150 lpi at 45 degreesOur investigation demonstrates that topical applica-tion of a-MSH could facilitate hyperemia and promotere-epitelization of the cornea in rats without significantside effects. However, the influence on the corneal epi-thelium healing was dose dependent with observed sta-tistically significant influence at a concentration of1×10–4 mg/mL of a-MSH. This is in accordance with thefact that a-MSH induced ocular hypotensive effect is alsodose dependent15,16 just as its activity in the nervoussystem18, skin19 and its influence as an anti-inflamma-tory agent20. The favorable hypotensive effect of a-MSHon intraocular pressure was achieved with aqueous phys-iological doses (10–8M – 10–9M)15. Our results are consis-tent with the data of Naveh et al.15,16 since we observedthat two most efficient doses with respect to the cornealwound healing ranged from 6×10–8M (1×10–3 mg/mL) to6×10–9 M (1×10–4 mg/mL). In some previous studiesa-MSH was used in higher non-physiological doses caus-ing flare and increased values of intraocular pressure af-ter its application21,22. Considering that these valueswere far greater than the physiological it is reasonable toconclude that we could benefit only if the doses close tothe physiological ones in the aqueous humor are ap-plied15,16. The possible topical application of a-MSHcould provide an advantage when compared to somelong-established but more aggressive methods that haveproven to facilitate corneal epithelial healing23.In summary, we have demonstrated that topical a--MSH medication could promote the healing of cornealwounds and simultaneously reduce the inflammatory re-sponse without the major side effects of corticosteroidtherapy. Further studies are therefore necessary to de-termine the most effective doses with the longest andmost permanent outcome with minimal relapses. Thuswe can suggest that a-MSH could be considered as a pos-sible future therapy for corneal defects.Conclusionsa-MSH concentration of 1×10–4 mg/mL significantlypromoted epithelial healing in rat corneas when com-pared to the control group, at 16 hours and 24 after epi-thelial abrasion.Compared to the animals treated with with 0.9%NaCl, a-MSH drops elicited a significant reduction of oc-ular irritation in all treated groups 16 hours after cornealabrasion.Topical a-MSH medication promotes the healing ofcorneal wounds and reduces local irritation and inflam-mation.AcknowledgementsThe support by the Ministry of Science, Educationand Sports, Republic of Croatia is thankfully acknowl-edged (Grant No. 098-0982929-2524).R E F E R E N C E S1. BRZOSKA T, LUGER TA, MAASER C, ABELS C, BÖHM M, En-docr Rev, 29 (2008) 581. DOI: 10.1210/er.2007-0027. — 2. GETTING SJ,Pharmacol Ther, 111 (2006) 1. DOI: 10.1016/j.pharmthera.2005.06.022.— 3. ZIANGIROVA GG, BOCHAROV VE, MALAEVA LV, OLINEVICHVB, SHERSTNEV VV, Vestn Oftalmol, 113 (1997) 26. — 4. LIPTON JM,CATANIA A, Immunol Today, 18 (1997) 140. DOI:10.1016/S0167-5699(97)01009-8 — 5. CUTULI M, CRISTIANI S, JAMES M, LIPTON JM,CATANIA A, J Leukoc Biol, 67 (2000) 233. — 6. KONJEVODA P, NASI]M, ]URKOVI] T, SIKIRI] P, SEIWERTH S, [TAMBUK N, Effects ofBPC 157 on the healing of corneal lesions. In: OHNO S, AOKI K, USUIM, UCHIO E (Eds) Uveitis Today (Esevier, Amsterdam, The Netherlands(1998). — 7. KONJEVODA P, [TAMBUK N, ARALICA G, POKRI] B, JPhysiol – Paris, 95 (2001) 277. DOI: 10.1016/S0928-4257(01)00038-9. —8. DRAIZE JH, WOODARDG, CALVERYH, J Pharmacol Exp Therap, 82(1944) 377. — 9. KLENKLER B, SHEARDOWN H, Exp Eye Res. 79(2004) 677. DOI: 10.1016/j.exer.2004.07.008. — 10. LU L, REINACH PS,KAOWW, Exp Biol Med, 226 (2001) 653. — 11. BARBA KR, SAMY A, LAIC, PERLMAN JI, BOUCHARD CS, J Cataract Refract Surg, 26 (2000)893. DOI: 10.1016/S0886-3350(00)00364-3. — 12. MCGHEE CN, DEANS, DANESH-MEYER H, Drug Saf, 25 (2002) 33. DOI: 10.2165/00002018-200225010-00004. — 13. TAYLOR AW, Chem Immunol Allergy, 92 (2007)71. — 14. ZIESKE JD, GIPSON IK, Agents that affect corneal woundhealing: modulation of structure and function. In: ALBERT DM, JAKO-BIEC FA (Eds) Principles and Practice of Ophthalmology end Basic Sci-ences (WB Saunders, Philadelphia, USA, 1994). — 15. NAVEH N, MAR-SHALL J, Graefes Arch Clin Exp Ophthalmol, 239 (2001) 840. DOI: 10.1007/s00417-001-0379-1. — 16. NAVEH N, KAPLAN-MESSAS A, MAR-SHALL J, Br J Ophthalmol, 84 (2000) 1411. DOI: 10.1136/bjo.84.12.1411.— 17. HAMRAH P, HASKOVA Z, TAYLOR AW, ZHANG Q, KSANDERBR, DANA MR, Transplantation, 88 (2009) 180. DOI: 10.1097/TP.0b013e3181ac11ea. — 18. CERIANI G, MACALUSO A, CATANIA A, LIPTONJM, Neuroendocrinology, 59 (1994) 138. DOI: 10.1159/000126650. — 19.HUNT G, Pathobiology, 63 (1995) 12. — 20. MACALUSO A, MCCOY D,CERIANI G, WATANABE T, BILTZ J, CATANIA A, LIPTON JM, J Neu-rosci, 14 (1994) 2377. — 21. HERNANDEZ DE, SIMONS KB, SPAMPI-NATO D, PEIFFER RLJ, DRAGO F, Neuropeptides, 6 (1985) 553. DOI:10.1016/0143-4179(85)90118-0. — 22. MCCULLEN RK, PEIFFER RL,JENNES L, HERNANDEZ DE, Neuropeptides, 12 (1988) 213. DOI: 10.1016/0143-4179(88)90057-1 — 23. DEKARIS I, MRAVICIC I, BARISICA, DRACA N, PAUK M, Coll Antropol, 34 (2010) Suppl 2 15.J. PavanUniversity of Zagreb, Dubrava University Hospital, Department of Ophthalmology, Avenija Gojka [u{ka 6,10000 Zagreb, Croatia.e-mail: josip.pavan@zg.htnet.hrJ. Pavan et al.: Alpha-MSH and Corneal Epithelial Lesions in Rats, Coll. Antropol. 36 (2012) 4: 1407–14111410U:\coll-antropolo\coll-antro-4-2012\10122 Pavan.vp21. prosinac 2012 11:31:04Color profile: DisabledComposite  150 lpi at 45 degreesDJELOVANJE a-MSH NA O[TE]ENJA EPITELA RO@NICE U [TAKORAS A @ E T A KIstra`ivali smo utjecaj alfa-melanostimuliraju}eg hormona (a-MSH) na cijeljenje epitelnih o{te}enja ro`nice u mu`-jaka Wistar {takora. Lezije su izazvane abrazijom epitela ro`nice kirur{kom lancetom pod kontrolom biomikroskopa.Prije abrazije epitel je omek{an djelovanjem 40% etanola. Primjena a-MSH kapi u koncentraciji od 1×10–4 mg/mLzna~ajno pospje{uje cijeljenje epitela u usporedbi s kontrolnom grupom lije~enom kapima 0,9% NaCl uz zna~ajno sma-njenje hiperemije oka. Lokalna primjena a-MSH mo`e pospje{iti cijeljenje rana ro`nice uz smanjenje lokalnog upalnogodgovora bez izra`enijih nuspojava kortikosteroidne terapije.J. Pavan et al.: Alpha-MSH and Corneal Epithelial Lesions in Rats, Coll. Antropol. 36 (2012) 4: 1407–14111411U:\coll-antropolo\coll-antro-4-2012\10122 Pavan.vp21. prosinac 2012 11:31:04Color profile: DisabledComposite  150 lpi at 45 degrees

Effects of Alpha-MSH on Corneal Epithelial Lesions in Rats

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Coll. Antropol. 36 (2012) 4: 1407–1411Original scientific paperEffects of Alpha-MSH on Corneal EpithelialLesions in RatsJosip Pavan1, Adrian Lukenda1, Nikola [tambuk2, Pa{ko Konjevoda2, Snje`ana Ka{telan1and Marko ]urkovi}11 University of Zagreb, Dubrava University Hospital, Department of Ophthalmology, Zagreb, Croatia.2 »Ru|er Bo{kovi}« Institute, Zagreb, CroatiaA B S T R A C TWe investigated the influence of alpha-melanocyte stimulating hormone (a-MSH) on the healing of corneal epitheliallesions in male Wistar rats. The lesions were induced by abrading corneal epithelium with a surgical lancet under abiomicroscope. Before the abrasion corneal epithelium was softened with 40% ethanol. a-MSH drops in a concentrationof 1×10–4 mg/mL significantly promoted epithelial healing, when compared to the control group treated with 0.9% NaCl.Significant reduction of ocular irritation was also observed. Topical a-MSH medication could promote the healing ofcorneal wounds and reduce local inflammatory response without the major side effects of the corticosteroid therapy.Key words: alpha-MSH, cornea, drops, epithelization, wound, healing, irritationIntroductionAlpha-melanocyte stimulating hormone (a-MSH) is apeptide deriving from the post-translational processingof the pro-opiomelanocortin (POMC) molecule1,2. ThePOMC prohormone gives rise to a variety of biologicallyactive peptides including adrenocorticotropin (ACTH)and a-MSH1,2. a-MSH corresponds to the first 13 aminoacids of the ACTH sequence. A synthetic peptide frag-ment of ACTH4–10 has been shown to improve the rabbitcornea wound healing3.Other results indicated that in addition to stronganti-inflammatory and cytoprotective effects on differentorgans and tissues a-MSH elicits antimicrobial and anti-fungal effects1,2,4,5.Impaired and prolonged reepithelization of corneal le-sions may result in long lasting corneal defects, whichcan possibly be followed by serious complications such asmicrobial and fungal inflammation and corneal opaci-fication. Therefore, we have investigated the effects ofa-MSH on the corneal healing and reduction of eye irri-tation caused by the abrasion of rat corneal epithelium.Materials and MethodsAnimalsWistar male albino rats weighing from 160 to 180 gwere used as the experimental animals. They were keptunder standard conditions with a 12 hour light/dark cy-cle. The rats were divided in groups of 6 animals and acontrol group had 8 animals.Corneal abrasionStandardized 3 mm wide circular lesions were in-duced by abrading corneal epithelium with a surgicallancet under a hand-held biomicroscope (Heine HSL100)6. Before scraping the cornea, epithelium was soft-ened with a circular filter paper soaked in 40% ethanol,placed on cornea for 30 seconds and irrigated with salinesolution. Abrasion procedure was done in light ether an-esthesia (Narkosaether, Lek, Slovenia). Immediately af-ter the abrasion, corneas were dyed with a sterile fluores-cein solution. High resolution digital camera (PanasonicNV-GX7, 1200000 pixels) was used to make the photo-graphs of abraded corneas under a cobalt blue UV lampat 0, 2, 8, 16 and 24 hour intervals.Treatment protocolCorneal lesions were treated with two eye drops ofa-MSH (SYSMEHFRWGKPV, >97% purity7 administer-ed in 10 seconds intervals, at 0, 2, 8 and 16 hours afterthe injury. The following eye drop concentrations wereused: 1 mg/mL, 1×10–1 mg/mL, 1×10–2 mg/mL, 1×10–31407Received for publication May 20, 2010U:\coll-antropolo\coll-antro-4-2012\10122 Pavan.vp21. prosinac 2012 11:31:04Color profile: DisabledComposite  150 lpi at 45 degreesmg/mL, 1×10–4 mg/mL. The control group was treatedwith 0.9% NaCl (1 drop = 0.05 mL).Evaluation and scoring of corneal lesionsand ocular irritationThe healing of corneal lesions was scored after the ini-tial abrasion using a modified Draize test6,8. Originally,Draize test is used to score the advancement of a lesionafter application of an irritant. In this experiment wehave reversed the method. The score was calculated asfollows: score 0 for no corneal lesion detected, score 1 for0–25%, score 2 for 25–50%, score 3 for 50–75% and score4 for 75–100% of initial lesion surface still not healed.The relative size of each lesion was compared to thewhole surface of the cornea using image processing soft-ware (ImageJ). The intensity of conjunctival irritationwas described by numbers, from 1 (for a minimal) to 10(for a maximal irritation as seen in saline treated eyes).The mean of the left and right eye score was used.Data analysisStatistical analysis and comparison of control andtreated groups was performed with Steel’s test – a non-parametric version of Dunett’s test. The analysis wasmade by using KyPlot software (version 4.0) and Graph-Pad Prism software (version 5.0).ResultsFive groups of experimental animals were treatedwith a-MSH drops in the following range of concentra-tions: 1 mg/mL, 1×10–1 mg/mL, 1×10–2 mg/mL, 1×10–3mg/mL and 1×10–4 mg/mL. The control group was treat-ed with 0.9% NaCl drops.a-MSH concentrations in the range from 1 mg/1 mLto 1?10–3 mg/mL did not significantly affect the rate ofcorneal healing (Table 1). The peptide concentration of1×10–4 mg/mL significantly promoted epithelial healingin rat corneas when compared to the control group, 16J. Pavan et al.: Alpha-MSH and Corneal Epithelial Lesions in Rats, Coll. Antropol. 36 (2012) 4: 1407–14111408TABLE 1EFFECTS OF DIFFERENT CONCENTRATIONS OF a-MSH ON THE HEALING OF CORNEAL EPITHELIAL DEFECTS 2, 8, 16 AND 24HOURS AFTER ABRASION (DESCRIPTIVE STATISTICS)Control (0.9% NaCl) N X Median Min Max Q1 Q3 SD Steel test2 h 8 99.8 101.4 60.6 140.6 87.1 109.9 24.28 h 8 68.9 70.1 22.3 92.6 65.0 83.0 21.516 h 8 43.7 38.2 16.8 84.0 29.3 56.9 22.624 h 8 16.3 14.0 2.7 49.0 7.6 17.9 14.2Alpha-MSH (1×10–4 mg/mL) N X Median Min Max Q1 Q3 SD p value2 h 6 75.7 72.5 53.4 110.5 70.6 74.8 18.8 0.6088 h 6 62.1 57.4 52.1 89.6 52.4 63.5 14.4 >0.99916 h 6 10.4 5.8 3.5 25.2 4.5 17.45 8.9 0.00824 h 6 0.1 0.0 0.0 0.6 0.3 0.6 0.2 0.007Alpha-MSH (1×10–3 mg/mL) N X Median Min Max Q1 Q3 SD p value2 h 6 78.5 78.8 58.8 99.6 68.1 86.7 14.2 >0.9998 h 6 66.9 70.9 42.7 85.3 56.4 75.3 15.3 >0.99916 h 6 24.9 23.5 7.6 42.1 10.1 38.5 14.2 >0.99924 h 6 1.2 0.0 0.0 5.2 0.00 3.7 2.1 0.001Alpha-MSH (1×10–2 mg/mL) N X Median Min Max Q1 Q3 SD p value2 h 6 85.4 85.8 69.4 103.5 76.4 91.4 12.1 >0.9998 h 6 57.0 58.2 43.3 68.4 51.9 62.2 8.7 >0.99916 h 6 20.2 19.8 8.2 36.8 8.5 27.9 11.1 0.69524 h 6 13.4 10.7 3.9 31.1 5.1 24.5 9.9 0.996Alpha-MSH (1×10–1 mg/mL) N X Median Min Max Q1 Q3 SD p value2 h 6 88.3 93.0 65.7 102.9 72.3 102.8 16.3 >0.9998 h 6 73.3 77.2 45.0 92.0 61.2 87.1 17.5 >0.99916 h 6 31.0 29.6 10.6 50.8 18.3 47.3 16.6 >0.99924 h 6 19.5 19.4 0.0 42.0 4.5 34.6 14.6 0.996Alpha-MSH (1mg/mL) N X Median Min Max Q1 Q3 SD p values2 h 6 9.0 113.8 73.4 138.1 95.3 119.7 22.2 >0.9998 h 6 75.7 77.9 50.6 91.1 74.5 82.1 13.7 >0.99916 h 6 24.6 24.3 13.9 41.3 14.9 28.8 10.2 >0.99924 h 6 17.3 18.7 5.2 25.6 7.8 25.4 8.2 0.853Alpha-MSH – alpha-melanocyte stimulating hormoneU:\coll-antropolo\coll-antro-4-2012\10122 Pavan.vp21. prosinac 2012 11:31:04Color profile: DisabledComposite  150 lpi at 45 degreesand 24 hours after epithelial abrasion (Table 1, Figure 1).a-MSH concentration of 1×10–3 mg/mL was also effec-tive 24 hours after epithelial abrasion (Table 1, Figure 1).Compared to the animals treated with 0.9% NaCl dropsa-MSH drops elicited a significant reduction of conjunc-tival hyperemia in all treated groups 16 hours after cor-neal abrasion (p<0.05, Table 2, Figure 2).DiscussionIntegrity of ocular surface epithelium and cornealre-epithelization is vital for normal hydration and trans-parency of the cornea. It may therefore have influence onvisual function and present a key process in preventionof corneal and intraocular infection9. During the healingprocess the migration and proliferation of the corneal ep-ithelium takes place. Simultaneously, activation of differ-ent growth factors as well as some cytokines occurs aspart of an inflammatory reaction10. Inflammatory andimmunological reactions inside the cornea may cause fi-brosis which could subsequently reduce corneal trans-parency and lead to blindness. Corticosteroids applied lo-cally have a beneficial effect on fibrosis prevention afterinjury. However, they also inhibit wound healing andthus can not be used until the integrity of the ocular sur-face is re-established. Moreover, they have a number ofknown side effects such as ocular hypertension, glau-coma, cataract formation, epithelial toxicity, crystallinekeratopathy, decreased wound strength and thereforethey should be applied very cautiously11,12.Although several substances have been reported toimprove the corneal healing rate6,12, it is still necessaryto search for substances that could facilitate cornealre-epithelization and prevent fibrosis at the same time.Besides the observed beneficial effects on corneal epithe-lization and irritation, a-MSH is known to play an im-portant role as a mediator of immunoregulatory andimmunosuppressory processes which occur in the aque-ous humor13. In anti-inflammatory reactions a-MSH isinvolved by suppression of prostaglandin E2 (PGE2), tu-mor necrosis factor-a (TNF-a), interleukin-1 (IL-1), in-terleukin-6 (IL-6) and monocyte chemotactic protein-1(MCP-1) production as well as by blocking cyclo-oxy-genase-2 (COX-2) expression14. Furthermore, it may alsobe useful as an anti-inflammatory agent in ocular trau-ma treatment maintaining blood-aqueous barrier integ-rity as opposed to steroids14. After penetrating corneo-limbal cut systemic and topical treatment with a-MSH inrabbits equal or better results in reducing intraocular in-flammatory reactions compared to topical application ofcorticosteroids were shown15. In the same animal modelits hypotensive ocular effect with sustained reduction ofintraocular pressure after a single topical applicationwas also established16. Moreover, the local subconjunc-tival treatment with a-MSH reduced allorejection of ratcorneal grafts17.J. Pavan et al.: Alpha-MSH and Corneal Epithelial Lesions in Rats, Coll. Antropol. 36 (2012) 4: 1407–14111409Contro l(0.9%NaCl)mg/mL )-4-MSH(1×10αmg/mL)-3-MSH(1×10αmg/mL)-2-MSH(1×10αmg/mL)-1-MSH(1×10αmg/mL )-MSH(1α0.02.55.07.510.012.515.017.520.022.525.027.5*** p < 0.05 vs. controlCornealepithelialdefectarea(%ofinitiallesion)Fig. 1. Healing of corneal defects – percentage of theinitial lesion after 24 hours. a-MSH – alpha-melanocytestimulating hormone.TABLE 2EFFECTS OF a-MSH ON OCULAR IRRITATION 16 HOURS AFTEREPITHELIAL ABRASION (INTENSITY SCORE FROM 1 FORMINIMAL TO 10 FOR MAXIMAL CONJUNCTIVAL HYPEREMIA)Substance X SD MedianSteeltest (p)Control 0.9% NaCl 9.7 1.22 10.0a-MSH 1 mg/mL 4.7 1.50 4.0 0.007a-MSH 1×10–4 mg/mL 5.2 1.47 5.5 0.007a-MSH 1×10–3 mg/mL 3.5 0.55 3.5 0.006a-MSH 1×10–2 mg/mL 5.2 0.98 5.5 0.007a-MSH 1×10–1 mg/mL 5.0 1.27 4.5 0.007a-MSH – alpha-melanocyte stimulating hormoneControl(0.9%NaCl)mg/mL)-4-MSH(1×10αmg/mL)-3-MSH(1×10αmg/mL)-2-MSH(1×10αmg/mL)-1-MSH(1×10α-MSH(1mg/mL)α012345678910**** ** p < 0.05 vs. controlEyeirritation(0-10)Fig. 2. Ocular irritation in a-MSH treated groups and con-trols 16 hours after epithelial abrasion. a-MSH – alpha-melano-cyte stimulating hormone.U:\coll-antropolo\coll-antro-4-2012\10122 Pavan.vp21. prosinac 2012 11:31:04Color profile: DisabledComposite  150 lpi at 45 degreesOur investigation demonstrates that topical applica-tion of a-MSH could facilitate hyperemia and promotere-epitelization of the cornea in rats without significantside effects. However, the influence on the corneal epi-thelium healing was dose dependent with observed sta-tistically significant influence at a concentration of1×10–4 mg/mL of a-MSH. This is in accordance with thefact that a-MSH induced ocular hypotensive effect is alsodose dependent15,16 just as its activity in the nervoussystem18, skin19 and its influence as an anti-inflamma-tory agent20. The favorable hypotensive effect of a-MSHon intraocular pressure was achieved with aqueous phys-iological doses (10–8 M – 10–9 M)15. Our results are consis-tent with the data of Naveh et al.15,16 since we observedthat two most efficient doses with respect to the cornealwound healing ranged from 6×10–8 M (1×10–3 mg/mL) to6×10–9 M (1×10–4 mg/mL). In some previous studiesa-MSH was used in higher non-physiological doses caus-ing flare and increased values of intraocular pressure af-ter its application21,22. Considering that these valueswere far greater than the physiological it is reasonable toconclude that we could benefit only if the doses close tothe physiological ones in the aqueous humor are ap-plied15,16. The possible topical application of a-MSHcould provide an advantage when compared to somelong-established but more aggressive methods that haveproven to facilitate corneal epithelial healing23.In summary, we have demonstrated that topical a--MSH medication could promote the healing of cornealwounds and simultaneously reduce the inflammatory re-sponse without the major side effects of corticosteroidtherapy. Further studies are therefore necessary to de-termine the most effective doses with the longest andmost permanent outcome with minimal relapses. Thuswe can suggest that a-MSH could be considered as a pos-sible future therapy for corneal defects.Conclusionsa-MSH concentration of 1×10–4 mg/mL significantlypromoted epithelial healing in rat corneas when com-pared to the control group, at 16 hours and 24 after epi-thelial abrasion.Compared to the animals treated with with 0.9%NaCl, a-MSH drops elicited a significant reduction of oc-ular irritation in all treated groups 16 hours after cornealabrasion.Topical a-MSH medication promotes the healing ofcorneal wounds and reduces local irritation and inflam-mation.AcknowledgementsThe support by the Ministry of Science, Educationand Sports, Republic of Croatia is thankfully acknowl-edged (Grant No. 098-0982929-2524).R E F E R E N C E S1. BRZOSKA T, LUGER TA, MAASER C, ABELS C, BÖHM M, En-docr Rev, 29 (2008) 581. DOI: 10.1210/er.2007-0027. — 2. GETTING SJ,Pharmacol Ther, 111 (2006) 1. DOI: 10.1016/j.pharmthera.2005.06.022.— 3. ZIANGIROVA GG, BOCHAROV VE, MALAEVA LV, OLINEVICHVB, SHERSTNEV VV, Vestn Oftalmol, 113 (1997) 26. — 4. LIPTON JM,CATANIA A, Immunol Today, 18 (1997) 140. DOI:10.1016/S0167-5699(97)01009-8 — 5. CUTULI M, CRISTIANI S, JAMES M, LIPTON JM,CATANIA A, J Leukoc Biol, 67 (2000) 233. — 6. KONJEVODA P, NASI]M, ]URKOVI] T, SIKIRI] P, SEIWERTH S, [TAMBUK N, Effects ofBPC 157 on the healing of corneal lesions. In: OHNO S, AOKI K, USUIM, UCHIO E (Eds) Uveitis Today (Esevier, Amsterdam, The Netherlands(1998). — 7. KONJEVODA P, [TAMBUK N, ARALICA G, POKRI] B, JPhysiol – Paris, 95 (2001) 277. DOI: 10.1016/S0928-4257(01)00038-9. —8. DRAIZE JH, WOODARD G, CALVERY H, J Pharmacol Exp Therap, 82(1944) 377. — 9. KLENKLER B, SHEARDOWN H, Exp Eye Res. 79(2004) 677. DOI: 10.1016/j.exer.2004.07.008. — 10. LU L, REINACH PS,KAO WW, Exp Biol Med, 226 (2001) 653. — 11. BARBA KR, SAMY A, LAIC, PERLMAN JI, BOUCHARD CS, J Cataract Refract Surg, 26 (2000)893. DOI: 10.1016/S0886-3350(00)00364-3. — 12. MCGHEE CN, DEANS, DANESH-MEYER H, Drug Saf, 25 (2002) 33. DOI: 10.2165/00002018-200225010-00004. — 13. TAYLOR AW, Chem Immunol Allergy, 92 (2007)71. — 14. ZIESKE JD, GIPSON IK, Agents that affect corneal woundhealing: modulation of structure and function. In: ALBERT DM, JAKO-BIEC FA (Eds) Principles and Practice of Ophthalmology end Basic Sci-ences (WB Saunders, Philadelphia, USA, 1994). — 15. NAVEH N, MAR-SHALL J, Graefes Arch Clin Exp Ophthalmol, 239 (2001) 840. DOI: 10.1007/s00417-001-0379-1. — 16. NAVEH N, KAPLAN-MESSAS A, MAR-SHALL J, Br J Ophthalmol, 84 (2000) 1411. DOI: 10.1136/bjo.84.12.1411.— 17. HAMRAH P, HASKOVA Z, TAYLOR AW, ZHANG Q, KSANDERBR, DANA MR, Transplantation, 88 (2009) 180. DOI: 10.1097/TP.0b013e3181ac11ea. — 18. CERIANI G, MACALUSO A, CATANIA A, LIPTONJM, Neuroendocrinology, 59 (1994) 138. DOI: 10.1159/000126650. — 19.HUNT G, Pathobiology, 63 (1995) 12. — 20. MACALUSO A, MCCOY D,CERIANI G, WATANABE T, BILTZ J, CATANIA A, LIPTON JM, J Neu-rosci, 14 (1994) 2377. — 21. HERNANDEZ DE, SIMONS KB, SPAMPI-NATO D, PEIFFER RLJ, DRAGO F, Neuropeptides, 6 (1985) 553. DOI:10.1016/0143-4179(85)90118-0. — 22. MCCULLEN RK, PEIFFER RL,JENNES L, HERNANDEZ DE, Neuropeptides, 12 (1988) 213. DOI: 10.1016/0143-4179(88)90057-1 — 23. DEKARIS I, MRAVICIC I, BARISICA, DRACA N, PAUK M, Coll Antropol, 34 (2010) Suppl 2 15.J. PavanUniversity of Zagreb, Dubrava University Hospital, Department of Ophthalmology, Avenija Gojka [u{ka 6,10000 Zagreb, Croatia.e-mail: josip.pavan@zg.htnet.hrJ. Pavan et al.: Alpha-MSH and Corneal Epithelial Lesions in Rats, Coll. Antropol. 36 (2012) 4: 1407–14111410U:\coll-antropolo\coll-antro-4-2012\10122 Pavan.vp21. prosinac 2012 11:31:04Color profile: DisabledComposite  150 lpi at 45 degreesDJELOVANJE a-MSH NA O[TE]ENJA EPITELA RO@NICE U [TAKORAS A @ E T A KIstra`ivali smo utjecaj alfa-melanostimuliraju}eg hormona (a-MSH) na cijeljenje epitelnih o{te}enja ro`nice u mu`-jaka Wistar {takora. Lezije su izazvane abrazijom epitela ro`nice kirur{kom lancetom pod kontrolom biomikroskopa.Prije abrazije epitel je omek{an djelovanjem 40% etanola. Primjena a-MSH kapi u koncentraciji od 1×10–4 mg/mLzna~ajno pospje{uje cijeljenje epitela u usporedbi s kontrolnom grupom lije~enom kapima 0,9% NaCl uz zna~ajno sma-njenje hiperemije oka. Lokalna primjena a-MSH mo`e pospje{iti cijeljenje rana ro`nice uz smanjenje lokalnog upalnogodgovora bez izra`enijih nuspojava kortikosteroidne terapije.J. Pavan et al.: Alpha-MSH and Corneal Epithelial Lesions in Rats, Coll. Antropol. 36 (2012) 4: 1407–14111411U:\coll-antropolo\coll-antro-4-2012\10122 Pavan.vp21. prosinac 2012 11:31:04Color profile: DisabledComposite  150 lpi at 45 degrees

Effects of Alpha-MSH on Corneal Epithelial Lesions in Rats

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