Ketorolac eye drops reduce inflammation and delay re-epithelization in response to corneal alkali burn in rabbits, without affecting iNOS or MMP-9

Colírio de cetorolaco reduz inflamação e atrasa a epitelização em resposta a queimadura por álcali em coelhos, sem afetar iNOS ou MMP-9

Tiago Barbalho Lima Alexandre Pinto Ribeiro Luciano Fernandes da Conceição Marcio Bandarra Wilson Gomez Manrique José Luiz Laus About the authors

Abstracts

Purposes:

To assess the effects of 0.5% ketorolac tromethamine without preservatives on the expression of iNOS and MMP-9 in alkali burn ulcers.

Methods:

Twelve eyes of 120-day-old male rabbits were treated (TG) every 6 h with 0.5% ketorolac tromethamine and 12 other eyes were treated with saline solution (CG), immediately after the occurrence of ulcers by 1 M sodium hydroxide (NaOH). Re-epithelialization was monitored using fluorescein every 6 h. After 24 h, six corneas (n=6) of each group were collected (M1). The others (n=6) were collected after reepithelialization (M2). At both moments, the inflammatory infiltrate and the conditions of the newly formed epithelium were histologically analyzed. iNOS and MMP-9 were evaluated by immunohistochemistry.

Results:

Mean epithelialization time in TG was 55 ± 0.84 h. In CG, it was 44 ± 1.06 h (p=0.001). At M1, corneas of TG had lower inflammatory exudation compared with (p <0.001). At M2, TG revealed discrete inflammatory exudation (p>0.05) and lower numbers of epithelial layers compared with CG. The mean iNOS in stromal cells did not differ in TG over both moments compared with CG (p>0.05) At M2, the central corneal region expressed more iNOS in both groups compared with the peripheral region. No significant differences were observed in iNOS scores of epithelial immunostaining between the groups and across M1 and M2 (p=0.69). Epithelial immunostaining scores for MMP-9 did not differ in TG compared with CG (p=0.69). The average immunostaining score of MMP-9 in stromal cells showed no differences between groups or moments. There was no correlation between immunostaining of iNOS and MMP-9 or between the amount of inflammatory cells and immunostaining of iNOS.

Conclusions:

Use of 0.5% keratolac tromethamine reduced inflammation and delayed reepithelialization in a cornea alkali burn model without impacting the expression of iNOS or MMP-9.

Corneal ulcer/chemically induced; Ketrolac tromethamine/administration; iNOS, MMP-9


Objetivos:

Avaliarem-se os efeitos do cetorolaco de trometamina 0,5%, sem conservante, sobre a expressão da iNOS e da MMP-9, em córneas com úlceras químicas.

Métodos:

Doze olhos de coelhos machos, 120 dias de idade, foram tratados (GT ), a cada 6 horas, com o cetorolaco de trometamina 0,5% e outros 12 com solução salina (GC), imediatamente à ocorrência de úlceras por hidróxido de sódio (NaOH) 1 mol/L. A reepitelização foi monitorada por fluresceína a cada seis horas. Decorridas 24 horas, seis córneas (n=6) de cada grupo foram colhidas (primeiro momento). As demais (n=6) o foram após a sua reepitelização (segundo momento). Em ambos os momentos, avaliaram-se o infiltrado inflamatório e as condições do epitélio neoformado (HE). Por imuno-histoquímica, avaliou-se a imunomarcação de iNOS e de MMP-9.

Resultados:

A média do tempo de epitelização no GT foi de 55 ± 0,84 horas. No GC, ela foi de 44 ± 1,06 horas (p=0,001). Às 24 horas, as córneas do GT apresentaram menor exsudação inflamatória (p<0,01). No segundo momento, o GT mostrou discreta exsudação inflamatória (p>0,05) e menor número de camadas epiteliais comparativamente ao GC. A média de imunomarcação de iNOS em células do estroma não diferiu do GT, em ambos os momentos (p>0,05). No segundo momento, a região central da córnea expressou mais iNOS, comparativamente à periférica, em ambos os grupos. Não se observaram diferenças significativas nos escores de imunomarcação epitelial de iNOS entre os grupos e os momentos (p=0,69). Os escores de imunomarcação epitelial para MMP-9 não diferiram entre os grupos (p=0,69). A média de imunomarcação da MMP-9 em células do estroma não exibiram diferenças entre os grupos e momentos da avaliação (p=0,32). Não houve correlação entre a imunomarcação de iNOS e de MMP-9, assim como quanto ao quantitativo de células inflamatórias e à imunomarcação de iNOS.

Conclusões:

Cetorolaco 0,5% reduziu a inflamação e atrasou a epitelização na queimadura corneal por álcali sem alterar a expressão de iNOS ou MMP-9 Descritores: Úlcera da córnea/induzida quimicamente; Cetorolaco de trometamina/administração; iNOS; MMP-9

Úlcera da córnea/induzida quimicamente; Cetorolaco de trometamina/administração; iNOS; MMP-9


INTRODUCTION

Non-steroidal anti-inflammatory drugs (NSAIDs) used in ulcerative keratitis may delay healing of epithelial lesions and enhance corneal stroma degradation(1Hendrix DV, Ward DA, Barnhill MA. Effects of anti-inflammatory drugs and preservatives on morphologic characteristics and migration of canine corneal epithelial cells in tissue culture. Vet Ophthalmol. 2002;5(2):127-35.,2Reviglio VE, Rana TS, Li QJ, Ashraf M,F, Daly MK, O'brien TP. Effects of topical nonsteroidal antinflammatory drugs on the expression of matrix metalloproteinases in the cornea. J Cataract Refract Surg. 2003;29(5):989-97.). An elevated expression of different metalloproteinases (MMPs) in the cornea locally treated with sodium diclofenac or ketorolac tromethamine has been demonstrated, even without preservatives(2Reviglio VE, Rana TS, Li QJ, Ashraf M,F, Daly MK, O'brien TP. Effects of topical nonsteroidal antinflammatory drugs on the expression of matrix metalloproteinases in the cornea. J Cataract Refract Surg. 2003;29(5):989-97.,3Ribeiro AP, Conceicao LF, Silva ML, Padua IR, Andrade AL, Luvizotto MC, et al. Effects of preservative free 0.5% ketorolac tromethamine in alkali burned rabbit corneas [abstract]. Vet Ophthalmol. 2010;13(5):360.).

Nitric oxide (NO) is a simple gaseous molecule found in small quantities in atmospheric air. It is synthesized with the help of nitric oxide synthase (NOS)(4Miranda KM, Espey MG, Jourd'heuil D, Grisham MB, Fukuto JM, Feelisch M, et al. The chemical biology of nitric oxide, In: Ignarro LJ, editor. Nitric oxide: biology and pathobiology. San Diego: Academic Press; 2000. p.41-55.). Three distinct forms of NOS have been recognized: two constitutive isoforms [nNOS (NOS-1) in the nervous system and eNOS (NOS-3) in endothelial cells] and the iNOS inducible isoform (NOS-2)(5Christopherson KS, Bredt DS. Nitric oxide in excitable tissues: physiological roles and diseases. J Clin Invest. 1997;100(10):2424-9.), which is expressed in different cells following transcriptional activation by cytokines or endotoxins(6Nathan C. Inducible nitric oxide synthase: what difference does it make? J Clin Invest. 1997;100(10):2417-23.). In ocular tissues, NO is involved in different events(7Becquet F, Courtois Y, Goureau O. Nitric oxide in the eye: multifaceted roles and diverse outcomes. Surv Ophthalmol. 1997;42(1):71-82.). In vitro, it has been demonstrated that stimulation of endothelial and bovine keratocyte cells by lipopolysaccharides and cytokines induces iNOS expression, releasing a large amount of NO(8Dighiero P, Behar-Cohen F, Courtois Y, Goureau O. Expression of inducible nitric oxide synthase in bovine corneal endothelial cells and keratocytes in vitro after lipopolysaccharide and cytokines stimulation. Invest Ophthalmol Vis Sci. 1997;38(10):2045-52.). iNOS expression in corneal lesions induced by ultraviolet radiation has been verified(9Chen BY, Lin DP, Wu CY, Teng MC, Sun CY, Tsai YT,et al. Dietary zerumbone prevents mouse cornea from UVB-induced photokeratitis through inhibition of NF-κB, iNOS, and TNF-α expression and reduction of MDA accumulation. Mol Vis. 2011;17:854-63.). In vivo, high levels of NO can be involved in corneal inflammatory diseases(8Dighiero P, Behar-Cohen F, Courtois Y, Goureau O. Expression of inducible nitric oxide synthase in bovine corneal endothelial cells and keratocytes in vitro after lipopolysaccharide and cytokines stimulation. Invest Ophthalmol Vis Sci. 1997;38(10):2045-52.). iNOS has been expressed in chemically ulcerated murine corneas, inhibiting neovascularization(1010 Sennlaub F, Courtois Y, Goureau O. Nitric oxide synthase-ii is expressed in severe corneal alkali burns and inhibits neovascularization. Invest Ophthalmol Vis Sci. 1999; 40(12):2773-9.). Peroxynitrite and NO reduce the levels of the tissue inhibitor of metalloproteinase-1 (TIMP-1), increasing the gelatinolytic activity of MMPs in corneal fibroblasts cultivated in vitro(1111 Brown DJ, Lin B, Chwa M, Atilano SR, Kim DW, Kenney MC. Elements of the nitric oxide pathway can degrade TIMP-1 and increase gelatinase activity. Mol Vis. 2004;10:281-8.).

MMP-9 is produced by epithelial cells and neutrophils and has a role in remodeling the corneal stroma after keratectomy(1212 Mulholland B, Tuft SJ, Khaw PT. Matrix metalloproteinase distribution during early corneal wound healing. Eye (London). 2005;19(5): 584-8.). MMP-9 is involved in corneal stroma degradation, facilitating the migration and proliferation of neo-vessels in ulcers(1212 Mulholland B, Tuft SJ, Khaw PT. Matrix metalloproteinase distribution during early corneal wound healing. Eye (London). 2005;19(5): 584-8.). iNOS expression has been studied in corneal ulcers caused by alkali(1010 Sennlaub F, Courtois Y, Goureau O. Nitric oxide synthase-ii is expressed in severe corneal alkali burns and inhibits neovascularization. Invest Ophthalmol Vis Sci. 1999; 40(12):2773-9.); however, the effect of NSAIDs on its activity is unknown.

Ketorolac tromethamine is used in ocular pain control, despite being toxic to the epithelium(3Ribeiro AP, Conceicao LF, Silva ML, Padua IR, Andrade AL, Luvizotto MC, et al. Effects of preservative free 0.5% ketorolac tromethamine in alkali burned rabbit corneas [abstract]. Vet Ophthalmol. 2010;13(5):360.), and is capable of raising MMPs expression(2Reviglio VE, Rana TS, Li QJ, Ashraf M,F, Daly MK, O'brien TP. Effects of topical nonsteroidal antinflammatory drugs on the expression of matrix metalloproteinases in the cornea. J Cataract Refract Surg. 2003;29(5):989-97.). In addition, its activity can be increased by iNOS, as demonstrated in vitro(1111 Brown DJ, Lin B, Chwa M, Atilano SR, Kim DW, Kenney MC. Elements of the nitric oxide pathway can degrade TIMP-1 and increase gelatinase activity. Mol Vis. 2004;10:281-8.).

The present study aimed to evaluate the effect of 0.5% ketorolac tromethamine without preservative on iNOS and MMP-9 expression in rabbit corneas with ulcers chemically induced by sodium hydroxide (NaOH).

METHODS

Male rabbits (average weight, 3.4 kg; age, 120 days) of the White New Zealand breed were selected [Oryctolagus cuniculus (Linnaeus, 1758)]. Animals were evaluated prior to the experiment using biomicroscopy with a slit lamp (XL-1 Slit lamp®; Shin-Nippon, Japan), applanation tonometry (Tono Pen XL®; Medtronic, Jacksonville, U.S.A.), indirect binocular ophthalmoscopy (Indirect Binocular ophthalmoscope FOH®; Eyetec S.A.), and fluorescein dye (Fluorescein 5 strips®-Ophthalmos Ltda., São Paulo/SP, Brazil). Healthy animals were individually housed in a ventilated environment, in appropriate, clean, and sanitized cages with a diet based on commercial food and drinking water ad libitum.

After induction of anesthesia by intramuscular injection of 15 mg/kg of ketamine [(S)-(+)-Ketamine®; Cristália, São Carlos/SP, Brazil] with 0.5 mg/kg midazolam (Dormire®; Cristália, São Carlos/SP, Brazil), periocular trichotomy was performed. A drop of proximetacaine was instilled (Anestalcon®; Alcon, São Paulo/SP, Brazil), and antiseptic treatment of the cornea, conjunctival sac, and conjunctivae were performed with iodine-polyvinylpyrrolidone (Laboriodine; Glicolabor Ind. Farmacêutica Ltda, Ribeirão Preto/SP, Brazil) diluted in saline (Sodium Chloride Solution 0.9%; JP Indústria Farmacêutica, Ribeirão Preto/SP, Brazil; 1:50). General anesthesia was achieved using masks with isoflurane (Forane®; Cristália, São Carlos/SP, Brazil), diluted in 100% oxygen in an open circuit.

After routine preparation of the operation field, a disk of filter paper (Whatman No. 40; F. Maia Ltda, Cotia/SP, Brazil), 6.0 mm in diameter, soaked in a solution of 1 M sodium hydroxide (NaOH) was gently maintained over the paracentral region of the cornea for 1 min. Immediately, careful washing of the entire corneal surface with saline solution 0.9% was performed.

Immediately after the lesions have been inflicted, 12 eyes were treated with 30 µL of 0.5% ketorolac tromethamine (Acular®; Allergan, Guarulhos/SP, Brazil), without preservatives (the treated group, TG), and 12 other eyes received, under similar regimen, 0.9% saline solution (control group, CG), both at regular intervals of 6 h, until the collection of corneas.

With lesions lasting for 24 h (M1, first moment), six corneas (n=6) from each group were collected. Remaining corneas (n=6) were collected after re-epithelialization (M2, second moment). Immediately, the preparation of samples for histology and immunohistochemistry was performed.

For monitoring re-epithelialization, clinical observations was performed using fluorescein dye tests and biomicroscopy with slit light and cobalt blue filter (Fluorescein in stripes®; Ophthalmos, São Paulo/SP, Brazil) every 6 h.

Histology

Corneas were processed and analyzed at the Laboratory of Immunohistochemistry at the Department of Veterinary Pathology, College of Agricultural and Veterinary Sciences, State University of São Paulo, Jaboticabal, SP, Brazil. The specimens were maintained in 10% buffered formalin for 24 h and then in 70% alcohol for 3 days. Next, samples were embedded in paraffin and cut sagittally in 5-µm sections. Hematoxylin-eosin (HE) was used for staining.

Using 40× light microscopy magnification (Olympus BX51; Olympus Optical Brazil, Ltda., São Paulo/SP, Brazil), the epithelial and conditions of the newly formed stroma were analyzed with regard to cellularity, disposition of collagen fibers, and inflammatory infiltration. Three random fields in the center of the corneas (adjacent to the lesion area) and three fields in the periphery (adjacent to the limbus) were analyzed.

Immunohistochemistry

Five-micrometer-thick sections were mounted on electrically charged slides and processed, employing the streptavidin-biotin complex technique, for labeling of iNOS and MMP-9. Sections were deparaffinized and hydrated in decreasing xylene batteries, followed by alcohol dehydration and rinsing in distilled water. Endogenous protein blocking was performed (Protein Block-DAKO® X0909). Retrieval of antigens was performed under heat and pressure (Pot Pascal-DAKO® S2800) in 10 mM buffered solution of sodium citrate with pH=6.0. Next, polyclonal primary antibody against iNOS (1:600) and MMP-9(1:200) were added. The material was incubated in a humid, dark chamber at a temperature of 23°C for 14 h. As secondary antibody, a polymer bound with peroxidase (ADVANCE-DAKO® K4069 HRP Rabbit/Mouse) was used, followed by incubation in a humid, dark chamber for 1 h. Visualization with diaminobenzidine chromogen (DAB; EnVision + System-HRP-DAKO®) was eventually performed. Counterstaining was performed using Harris’s hematoxylin. For the negative control, only the primary antibody was excluded from the reaction. Slides were washed three times for 5 min using PBS (pH=7.4).

For quantification, average values of three random fields were calculated during evaluation of epithelial immunostaining, using a semiquantitative index(1313 Stern NE, Gao J, Beuerman RW, Farley W, Zhuo L, McDonnell PJ, et al. Effects of fourth-generation fluorquinolones on the ocular surface, epithelium, and wound healing. Cornea. 2006;25(9 Suppl 2):S12-24.). For stromal immunostaining, three random fields in the central region (adjacent to the lesion area) and three fields in the peripheral region (adjacent to the limbus) were counted.

Statistical analysis

Data were evaluated for normality employing KolmogorovSmirnov’s test. Variance analysis was used for repeated measurements, and further analyses were conducted using Bonferroni’s and Tukey’s tests. Correlations between immunostaining scores of iNOS and MMP-9 and between iNOS and the amount of inflammatory cells were analyzed using Pearson’s correlation test. A minimum significance level of p<0.05 was used. It was considered that variables showed weak correlation when p<0.05, moderate correlation when p<0.01, and strong correlation when p<0.001. Results were expressed as means and standard error of the mean.

RESULTS

Average epithelialization time was 55 ± 0.84 h (second time) in TG and 44 ± 1.06 h in CG (p=0.001).

Histology

Sections stained with Hematoxylin-Eosin staining exhibited areas denuded of epithelium; this was more extensive in corneas treated with 0.5% ketorolac tromethamine without preservative than those of CG after 24 h. The newly formed epithelium in TG was largely characterized by a single layer. Control corneas showed newly formed epithelium, with alternating areas of single and two or more layers.

At M2, the treated corneas proved to be epithelialized, sometimes exhibiting areas of epithelium in double layers still not adhering in the center of the lesion. In the same period, control corneas revealed a higher stratification.

Regarding stromal conditions, in both groups and at both moments, low cellularity, collagen fiber disorganization, and edema were observed.

Regarding inflammatory cellularity, control corneas exhibited margination and diapedesis of polymorphonuclear cells, with a predominance of neutrophils and with mononuclear cells being rare. After 55 h, a mild inflammatory exudate was noticed in the treated corneas, with rare polymorphonuclear and inflammatory mononuclear cells compared with controls. The amount of inflammatory cells in the corneal periphery at M1, was significantly lower in the TG (395.5 ± 40.97) compared with the CG (1644 ± 246.6; p<0.001). Values were also smaller in the central region of TG (159.7 ± 25.06) at 24 h compared with the CG (474.8 ± 104.3; p>0.05). Moreover, at M1, the amount of inflammatory cells in the periphery of control corneas (1644 ± 246.6) was significantly higher compared with that in the central area (474.8 ± 104.3 cells; p<0.001; Figure 1). At M2, the amount of inflammatory cells in the periphery was lower in TG (430.5 ± 71.41) compared with CG (960.3 ± 170.3). In the central region, similar results were found in TG (276.3 ± 30.11) and CG (277.8 ± 40.53; Figure 1). The number of cells in the corneal periphery of CG significantly decreased between M1 and M2 (p<0.01; Figure 1).

Figure 1
Mean and standard error* of numbers of inflammatory cells in the periphery (P) and center (C) of male adult rabbit corneas ulcerated with 1 M NaOH and treated locally with 0.5% ketorolac tromethamine without preservative (gray) or with 0.9% saline solution (white) at first moment (M1, 24 hours after chemical burn) and second moment (M2, after reepithelialization). *Bonferroni’s Test.

Immunohistochemistry

iNOS immunostaining of the epithelium was conspicuously more intense in the margins of the lesions. Regarding the stroma, immunostaining of the cytoplasm and of the nuclei of polymorphonuclear and mononuclear cells and keratocytes was observed to be more intense in the region adjacent to the lesion (Figure 2).

Figure 2
Photomicrographs of iNOS immunostaining of male adult rabbit corneas ulcerated with 1 M NaOH and locally treated with 0.5% tromethamine ketorolac (A and B) without preservative or (C and D) with 0.9% saline solution. A) Immunostained cells (arrows) in the corneal stroma treated at first moment (M1, 24 hours after chemical burn). B) Immunostained cells (arrows) in the corneal stroma treated at second moment (M2, after reepithelialization). C) Immunostained cells (arrows) in the control corneal stroma treated at M1. D) Immunostained cells (arrows) in the control corneal stroma at M2. Diaminobenzidine. (Scale bar=50 µm).

At M1, iNOS expression in the corneal epithelium was similar between the two groups. At M2, a non-significant lower expression was observed in TG (1.5 ± 0.22) compared with CG (2 ± 0.44; p=0.69; Figure 3).

Figure 3
Mean and standard error* of quantitative iNOS immunostaining of stromal cells in the periphery (P) and center (C) of male adults rabbit corneas ulcerated with 1 M NaOH and treated locally with 0.5% ketorolac tromethamine without preservative (gray) or with 0.9% saline solution (white) at first moment (M1, 24 hours after chemical burn) and second moment (M2, after reepithelialization). *Bonferroni’s Test.

When the periphery and the stroma center were analyzed at M1, no difference in iNOS expression in the treated corneas was observed. At M2, a significantly higher expression was found in the central region (48.83 ± 9.93) of treated corneas compared with the peripheral region (14.5 ± 2.88) in the same group (p<0.01). iNOS expression in the central region (45.5 ± 7.43) in control corneas was significantly higher compared with the peripheral region (12.67 ± 5.07) at M2 (p<0.05; Figure 3). At M1, there was no difference between the regions in the two groups.

Regarding MMP-9, immunostaining was predominantly evident in the cytoplasm of epithelial cells (Figure 4). MMP-9 expression was observed in smaller quantity in keratocytes and in polymorphonuclear and in mononuclear cells.

Figure 4
Photomicrographs of MMP immunostaining in male adult rabbit corneas ulcerated with 1 M NaOH and locally treated with 0.5% ketorolac tromethamine (A and B) without preservative or (C and D) with 0.9% saline solution. A. Immunostaining of the corneal epithelium (arrows) treated at first moment (M1, 24 hours after chemical burn). B. Immunostaining of the corneal epithelium (arrows) treated at second moment (M2, after reepithelialization). C. Immunostaining (arrows) of the control cornea at M1. D. Immunostaining of the epithelium (arrows) of the control cornea at M2. Diaminobenzidine (Bar=50 µm).

When assessing immunostaining scores in the epithelium of treated corneas at M1, a lower MMP-9 expression was observed (1.16 ± 0.16) compared with CG (1.5 ± 0.34). At M2, MMP-9 expression in the epithelium of treated corneas was lower (1.5 ± 0.3) compared with the expression observed in CG (1.5 ± 0.22); however, this difference was not statistically significant (p=0.69).

Regarding average quantity of immunostained cells at M1, MMP-9 expression was lower in the periphery of treated corneas (7 ± 1.98) compared with control corneas (23.5 ± 7.34; p<0.001). In the central region, there were negligible differences between TG (24 ± 9.45) and CG (26.5 ± 8.92). At M2, the mean quantity of immunostained cells in the periphery of treated (9.16 ± 4.24) and control (9 ± 3.97) corneas did not differ. In the central area, there was a higher expression in TG(22.83 ± 13.41) than in CG (15.5 ± 6.52). No significant difference was found when comparing the values (p=0.32).

No correlation was observed between iNOS and MMP-9 in the epithelium of TG (r=0.30 and p=0.34) and in the stroma of treated (r=0.20 and p=0.54) and CG (r=0.41 and p=0.18). Moderate correlation (r=0.70 and p=0.01) was observed between iNOS and MMP-9 expression in the epithelium of CG.

No correlation was observed between iNOS expression and the amount of inflammatory cells in the stroma in TG (r=0.28 and p=0.38) or CG (r=0.56 and p=0.06).

DISCUSSION

Repeated instillation of ketorolac tromethamine influences the development of corneal lesions(1414 Pereira AE, Silva MR, Froes RC, Marques MA. Regeneracao do epitelio corneano com uso topico de cetorolac de trometamina. Rev Bras Oftalmol. 2001;60(10):695-701.) as also evidenced by the results found in this study. The commercial formulation, i.e., without preservatives, was chosen because it may be toxic to the epithelium(1515 Guo Y, Satpathy M, Wilson G, Srinivas SP. Benzalkonium chloride induces dephosphorylation of myosin light chain in cultured corneal epithelial cells. Invest Ophthalmol Vis Sci. 2007;48(5):2001-8.,1616 Epstein SP, Ahdoot M, Marcus E, Asbell P. Comparative toxicity of preservatives on immortalized corneal and conjunctival epithelial cells. J Ocul Pharmacol Ther. 2009; 25(2):113-9.). Control corneas epithelialized faster, and this observation supports the fact that the used drug was toxic and the effect could have resulted from changes in the cytoskeleton of epithelial cells(1717 Agrawal VB, Tsai RJ. Corneal epithelium wound healing. Indian J Ophtahlmol. 2003; 51(1):5-15.). Similarly, desquamation and decrease in microvilli in corneas treated with ketorolac tromethamine without preservatives have been observed(3Ribeiro AP, Conceicao LF, Silva ML, Padua IR, Andrade AL, Luvizotto MC, et al. Effects of preservative free 0.5% ketorolac tromethamine in alkali burned rabbit corneas [abstract]. Vet Ophthalmol. 2010;13(5):360.).

The effect of ketorolac tromethamine and its preservatives on the epithelialization of debrided corneas with 95% ethyl alcohol has been studied in rabbits(1414 Pereira AE, Silva MR, Froes RC, Marques MA. Regeneracao do epitelio corneano com uso topico de cetorolac de trometamina. Rev Bras Oftalmol. 2001;60(10):695-701.). The authors found that eyes treated with NSAIDs were still ulcerated 60 h after abrasion. In the present study, treated eyes had epithelialized by 55 h following abrasion.

The present investigation showed significantly lower inflammatory exudation in treated eyes. Similar findings were presented after using ketorolac tromethamine with preservatives in chemical ulcers in rabbits(1414 Pereira AE, Silva MR, Froes RC, Marques MA. Regeneracao do epitelio corneano com uso topico de cetorolac de trometamina. Rev Bras Oftalmol. 2001;60(10):695-701.). Infiltration by polymorphonuclear leukocytes, mainly neutrophils, is necessary for epithelialization. The inflammatory infiltrate influences resident cells by producing cytokines that modulate tissue repair(1818 Gan L, Fagerholm P, Kim H. Effect of leukocytes on corneal cellular proliferation and wound healing. Invest Ophthalmol Vis Sci. 1999;40(3):575-81.).

The number of inflammatory cells in the periphery was higher in control corneas. The amount of cells in the central region did not significantly differ between the groups. In a sterile inflammation model, central infiltrates consist mainly of neutrophils and monocytes(1010 Sennlaub F, Courtois Y, Goureau O. Nitric oxide synthase-ii is expressed in severe corneal alkali burns and inhibits neovascularization. Invest Ophthalmol Vis Sci. 1999; 40(12):2773-9.); this in agreement with the findings of this study.

It has been demonstrated that iNOS is expressed in rat corneas during the inflammatory course following chemical abrasion by alkali(1010 Sennlaub F, Courtois Y, Goureau O. Nitric oxide synthase-ii is expressed in severe corneal alkali burns and inhibits neovascularization. Invest Ophthalmol Vis Sci. 1999; 40(12):2773-9.). The expression reached a maximum between 4 and 7 days following cauterization in the central corneal area. These data were consistent the findings of this study, in which the greatest number of positive iNOS cells occurred in M2 of the evaluation; in the central region, despite a higher number of inflammatory cells in the periphery.

Immunostaining of iNOS were similar to that observed for neutrophils(1010 Sennlaub F, Courtois Y, Goureau O. Nitric oxide synthase-ii is expressed in severe corneal alkali burns and inhibits neovascularization. Invest Ophthalmol Vis Sci. 1999; 40(12):2773-9.). However, few cells showed positive labeling for macro-phages. These observations suggest that neutrophils and monocytes may be the primary source of NO in corneas. Using the same model of chemical abrasion and considering the type of inflammatory infiltrate found in the corneas in this study, the same cells are speculated to be involved in iNOS expression. However, no correlation between iNOS expression and the amount of inflammatory cells was found. Langerhans cells have been found in the central epithelium after thermal cauterization(1919 Williamson JS, Dimarco S, Streilein JW. Immunobiology of Langerhans cells on the ocular surface. Invest Ophthalmol Vis Sci. 1987;28(9):1527-32.) and they may act in NO production.

Immunostaining of iNOS in keratocytes was observed in areas adjacent to the lesions. In vitro, iNOS may be expressed by keratocytes upon stimulation by cytokines(8Dighiero P, Behar-Cohen F, Courtois Y, Goureau O. Expression of inducible nitric oxide synthase in bovine corneal endothelial cells and keratocytes in vitro after lipopolysaccharide and cytokines stimulation. Invest Ophthalmol Vis Sci. 1997;38(10):2045-52.). The possibility that these cells have contributed to in vitro production of NO could not be excluded. Reactions that occur in different stages of corneal repair, however, are complex compared with those processed in vitro(1010 Sennlaub F, Courtois Y, Goureau O. Nitric oxide synthase-ii is expressed in severe corneal alkali burns and inhibits neovascularization. Invest Ophthalmol Vis Sci. 1999; 40(12):2773-9.).

iNOS expression was higher when compared with controls but the difference was nonsignificant. Contrary to these results, a proangiogenic role of NO in vitro has been reported(2020 Ziche M, Morbidelli L, Masini E, Amerini S, Granger HJ, Maggi CA, et al. Nitric oxide mediates angiogenesis in vivo and endothelial cell growth and migration in vitro promoted by substance P. J Clin Invest. 1994;94(5):2036-44.,2121 Ziche M, Morbidelli L, Choudhuri R, Zhang HT, Donnini S, Granger HJ, et al. Nitric oxide synthase lies downstream from vascular endothelial growth factor-induced but not basic fibroblast growth factor-induced angiogenesis. J Clin Invest. 1997;99(11):2625-34.). Such differences may be related with the expression of distinct isoforms of NOS dependent on the experimental model employed. The mechanism by which NO inhibits angiogenesis in the corneal cauterization model is not completely understood. However, it is clear that inhibition of angiogenesis by iNOS decreases the amount of inflammatory cells by inducing apoptosis(2222 Kim JC, Park GS, Kim JK, Kim YM. The role of nitric oxide in ocular surface cells. J Korean Med Sci. 2002;17(3):389-94.). When inhibiting inflammatory exudation, NSAIDs decrease neovascularization, with iNOS playing an important role. Involvement of other NOS isoforms, except iNOS, in vasculature induction has been demonstrated(9Chen BY, Lin DP, Wu CY, Teng MC, Sun CY, Tsai YT,et al. Dietary zerumbone prevents mouse cornea from UVB-induced photokeratitis through inhibition of NF-κB, iNOS, and TNF-α expression and reduction of MDA accumulation. Mol Vis. 2011;17:854-63.). These findings justify the expression of iNOS in TG and the lack of correlation between an event and the amount of inflammatory cells.

Kim et al. studied iNOS immunostaining after laser-assisted lamellar keratoplasty(2222 Kim JC, Park GS, Kim JK, Kim YM. The role of nitric oxide in ocular surface cells. J Korean Med Sci. 2002;17(3):389-94.). Chen et al. observed iNOS immunostaining in corneal epithelium upon stimulation by ultraviolet radiation(9Chen BY, Lin DP, Wu CY, Teng MC, Sun CY, Tsai YT,et al. Dietary zerumbone prevents mouse cornea from UVB-induced photokeratitis through inhibition of NF-κB, iNOS, and TNF-α expression and reduction of MDA accumulation. Mol Vis. 2011;17:854-63.). In the present study, iNOS expression was similar in both groups, despite the study drug being toxic to the epithelium.

Regarding MMP-9, we observed pronounced immunostaining in the epithelial area adjacent to the lesion. Authors have reported that basal epithelial cells synthesized MMP-9 in corneas that underwent lamellar keratectomy(2323 Ye HQ, Azar DT. Expression of gelatinases A and B, and TIMPs 1 and 2 during corneal wound healing. Invest Ophthalmol Vis Sci. 1998;39(6):913-21.); in addition, inflammatory cells and keratocytes were positive by immunostaining for MMP-9.MMP-9 is involved in the early stages of corneal epithelium repair(2323 Ye HQ, Azar DT. Expression of gelatinases A and B, and TIMPs 1 and 2 during corneal wound healing. Invest Ophthalmol Vis Sci. 1998;39(6):913-21.,2424 Azar DT, Hahn TW, Jain S, Yeh YC, Stetler-Stevenson SW. Matrix metalloproteinases are expressed during wound healing after excimer laser keratectomy. Cornea. 1996; 15(1):18-24.). In the present study, MMP-9 expression was observed in the epithelium at both M1 and M2. When stromal cells were evaluated, a higher expression in the corneal central region was observed at M1. The highest activity of MMP-9 was observed at M1, which was also observed in cases of keratotomy(2323 Ye HQ, Azar DT. Expression of gelatinases A and B, and TIMPs 1 and 2 during corneal wound healing. Invest Ophthalmol Vis Sci. 1998;39(6):913-21.).

Changes in MMPs due to the local use of NSAIDs may delay corneal epithelium repair(2525 O'Brien TP, Li QJ, Sauerburger F, Reviglio V E, Rana T, Ashraf MF. The role of matrix Metalloproteinases in ulcerative keratolysis associated with perioperative diclofenac use. Ophthalmology. 2001;108(4):656-9.). Collagenases may impair cicatricial healing, breaking the basal membrane interactions of epithelial cells(2525 O'Brien TP, Li QJ, Sauerburger F, Reviglio V E, Rana T, Ashraf MF. The role of matrix Metalloproteinases in ulcerative keratolysis associated with perioperative diclofenac use. Ophthalmology. 2001;108(4):656-9.). MMP production was found in the intact corneal epithelium after NSAIDs instillation(2Reviglio VE, Rana TS, Li QJ, Ashraf M,F, Daly MK, O'brien TP. Effects of topical nonsteroidal antinflammatory drugs on the expression of matrix metalloproteinases in the cornea. J Cataract Refract Surg. 2003;29(5):989-97.). Such findings indicate their involvement in the worsening of corneal ulcers(2Reviglio VE, Rana TS, Li QJ, Ashraf M,F, Daly MK, O'brien TP. Effects of topical nonsteroidal antinflammatory drugs on the expression of matrix metalloproteinases in the cornea. J Cataract Refract Surg. 2003;29(5):989-97.,2525 O'Brien TP, Li QJ, Sauerburger F, Reviglio V E, Rana T, Ashraf MF. The role of matrix Metalloproteinases in ulcerative keratolysis associated with perioperative diclofenac use. Ophthalmology. 2001;108(4):656-9.).

Delayed corneal repair associated with the use of local NSAIDs and expression of MMP-1, MMP-8, MMP-2 at significantly high levels, and low levels of MMP-9 in corneas treated with ketorolac tromethamine have been reported(2Reviglio VE, Rana TS, Li QJ, Ashraf M,F, Daly MK, O'brien TP. Effects of topical nonsteroidal antinflammatory drugs on the expression of matrix metalloproteinases in the cornea. J Cataract Refract Surg. 2003;29(5):989-97.). In this study, lower expression of MMP-9 was observed in the epithelium of TG compared with CG, but without significant differences. Other authors found no difference in the expression of MMPs; however, differences in the amount of MMP-9 and MMP-2 were determined by zymography(2626 Ribeiro AP, Silva ML, Araujo RL, Ferrucci DL, Mineo T, Thiesen R, et al. Expression of matrix metalloproteinases, type IV collagen, and interleukin-10 in rabbits treated with morphine after lamellar keratectomy. Vet Ophthalmol. 2012;15(3):153-63.).

In inflammatory processes, it has been demonstrated that NO and peroxynitrite can alter levels of MMPs and tissue inhibitors of MMPs (TIMPs)(2727 Hirai Y, Migita K, Honda S, Ueki Y, Yamasaki S, Urayama S, et al. Effects of nitric oxide on matrix metalloproteinase-2 production by rheumatoid synovial cells. Life Sci. 2001;68(8):913-20.). However, no correlation between MMP-9 expression and iNOS was found in this study. Data are available on the involvement of oxidative stress in inflammatory processes, in which macrophages, polymorphonuclear cells, and other inflammatory cells are involved in the activation of lytic enzymes(1111 Brown DJ, Lin B, Chwa M, Atilano SR, Kim DW, Kenney MC. Elements of the nitric oxide pathway can degrade TIMP-1 and increase gelatinase activity. Mol Vis. 2004;10:281-8.). High concentrations of peroxynitrite in vitro reduced TIMP-1 and hence the inhibition of MMP-9(2828 Frears ER, Zhang Z, Blake DR, O'connell JP, Winyard PG. Inactivation of tissue inhibitor of metalloproteinase-1 by peroxynitrite. FEBS Lett. 1996;381(1-2):21-4.). Similar results were found by Brown et al.(1111 Brown DJ, Lin B, Chwa M, Atilano SR, Kim DW, Kenney MC. Elements of the nitric oxide pathway can degrade TIMP-1 and increase gelatinase activity. Mol Vis. 2004;10:281-8.) in 2004, showing a cellular response to oxidative stress induced by peroxynitrite, suggesting a relation between elements of oxidative stress with activity of degradative enzymes. Other MMPs may be activated and involved in the reactions, as would be with regard to the higher activity of TIMPs.

Based on the obtained results, 0.5% ketorolac tromethamine without preservative may induce less inflammatory exudation and delay corneal epithelialization, eliciting non-significant changes in the expression of iNOS and MMP-9.

  • Funding: FAPESP process number 2011/07848-0 and CNPq process number 300833/2010-5
  • Approved by the following Research Ethics Committee on Animal Use: Comissão de Ética do Uso de Animais (CEUA) - UNESP (protocol number 007616/11).

ACKNOWLEDGEMENTS

The authors are thankful to the Fundação de Amparo à Pesquisa of the State of São Paulo-FAPESP (proc. 2011/07848-0) and to the Conselho Nacional de Desenvolvimento Científico e tecnológico - CNPq (proc. 300833/2010-5) for financial support.

REFERENCES

  • 1
    Hendrix DV, Ward DA, Barnhill MA. Effects of anti-inflammatory drugs and preservatives on morphologic characteristics and migration of canine corneal epithelial cells in tissue culture. Vet Ophthalmol. 2002;5(2):127-35.
  • 2
    Reviglio VE, Rana TS, Li QJ, Ashraf M,F, Daly MK, O'brien TP. Effects of topical nonsteroidal antinflammatory drugs on the expression of matrix metalloproteinases in the cornea. J Cataract Refract Surg. 2003;29(5):989-97.
  • 3
    Ribeiro AP, Conceicao LF, Silva ML, Padua IR, Andrade AL, Luvizotto MC, et al. Effects of preservative free 0.5% ketorolac tromethamine in alkali burned rabbit corneas [abstract]. Vet Ophthalmol. 2010;13(5):360.
  • 4
    Miranda KM, Espey MG, Jourd'heuil D, Grisham MB, Fukuto JM, Feelisch M, et al. The chemical biology of nitric oxide, In: Ignarro LJ, editor. Nitric oxide: biology and pathobiology. San Diego: Academic Press; 2000. p.41-55.
  • 5
    Christopherson KS, Bredt DS. Nitric oxide in excitable tissues: physiological roles and diseases. J Clin Invest. 1997;100(10):2424-9.
  • 6
    Nathan C. Inducible nitric oxide synthase: what difference does it make? J Clin Invest. 1997;100(10):2417-23.
  • 7
    Becquet F, Courtois Y, Goureau O. Nitric oxide in the eye: multifaceted roles and diverse outcomes. Surv Ophthalmol. 1997;42(1):71-82.
  • 8
    Dighiero P, Behar-Cohen F, Courtois Y, Goureau O. Expression of inducible nitric oxide synthase in bovine corneal endothelial cells and keratocytes in vitro after lipopolysaccharide and cytokines stimulation. Invest Ophthalmol Vis Sci. 1997;38(10):2045-52.
  • 9
    Chen BY, Lin DP, Wu CY, Teng MC, Sun CY, Tsai YT,et al. Dietary zerumbone prevents mouse cornea from UVB-induced photokeratitis through inhibition of NF-κB, iNOS, and TNF-α expression and reduction of MDA accumulation. Mol Vis. 2011;17:854-63.
  • 10
    Sennlaub F, Courtois Y, Goureau O. Nitric oxide synthase-ii is expressed in severe corneal alkali burns and inhibits neovascularization. Invest Ophthalmol Vis Sci. 1999; 40(12):2773-9.
  • 11
    Brown DJ, Lin B, Chwa M, Atilano SR, Kim DW, Kenney MC. Elements of the nitric oxide pathway can degrade TIMP-1 and increase gelatinase activity. Mol Vis. 2004;10:281-8.
  • 12
    Mulholland B, Tuft SJ, Khaw PT. Matrix metalloproteinase distribution during early corneal wound healing. Eye (London). 2005;19(5): 584-8.
  • 13
    Stern NE, Gao J, Beuerman RW, Farley W, Zhuo L, McDonnell PJ, et al. Effects of fourth-generation fluorquinolones on the ocular surface, epithelium, and wound healing. Cornea. 2006;25(9 Suppl 2):S12-24.
  • 14
    Pereira AE, Silva MR, Froes RC, Marques MA. Regeneracao do epitelio corneano com uso topico de cetorolac de trometamina. Rev Bras Oftalmol. 2001;60(10):695-701.
  • 15
    Guo Y, Satpathy M, Wilson G, Srinivas SP. Benzalkonium chloride induces dephosphorylation of myosin light chain in cultured corneal epithelial cells. Invest Ophthalmol Vis Sci. 2007;48(5):2001-8.
  • 16
    Epstein SP, Ahdoot M, Marcus E, Asbell P. Comparative toxicity of preservatives on immortalized corneal and conjunctival epithelial cells. J Ocul Pharmacol Ther. 2009; 25(2):113-9.
  • 17
    Agrawal VB, Tsai RJ. Corneal epithelium wound healing. Indian J Ophtahlmol. 2003; 51(1):5-15.
  • 18
    Gan L, Fagerholm P, Kim H. Effect of leukocytes on corneal cellular proliferation and wound healing. Invest Ophthalmol Vis Sci. 1999;40(3):575-81.
  • 19
    Williamson JS, Dimarco S, Streilein JW. Immunobiology of Langerhans cells on the ocular surface. Invest Ophthalmol Vis Sci. 1987;28(9):1527-32.
  • 20
    Ziche M, Morbidelli L, Masini E, Amerini S, Granger HJ, Maggi CA, et al. Nitric oxide mediates angiogenesis in vivo and endothelial cell growth and migration in vitro promoted by substance P. J Clin Invest. 1994;94(5):2036-44.
  • 21
    Ziche M, Morbidelli L, Choudhuri R, Zhang HT, Donnini S, Granger HJ, et al. Nitric oxide synthase lies downstream from vascular endothelial growth factor-induced but not basic fibroblast growth factor-induced angiogenesis. J Clin Invest. 1997;99(11):2625-34.
  • 22
    Kim JC, Park GS, Kim JK, Kim YM. The role of nitric oxide in ocular surface cells. J Korean Med Sci. 2002;17(3):389-94.
  • 23
    Ye HQ, Azar DT. Expression of gelatinases A and B, and TIMPs 1 and 2 during corneal wound healing. Invest Ophthalmol Vis Sci. 1998;39(6):913-21.
  • 24
    Azar DT, Hahn TW, Jain S, Yeh YC, Stetler-Stevenson SW. Matrix metalloproteinases are expressed during wound healing after excimer laser keratectomy. Cornea. 1996; 15(1):18-24.
  • 25
    O'Brien TP, Li QJ, Sauerburger F, Reviglio V E, Rana T, Ashraf MF. The role of matrix Metalloproteinases in ulcerative keratolysis associated with perioperative diclofenac use. Ophthalmology. 2001;108(4):656-9.
  • 26
    Ribeiro AP, Silva ML, Araujo RL, Ferrucci DL, Mineo T, Thiesen R, et al. Expression of matrix metalloproteinases, type IV collagen, and interleukin-10 in rabbits treated with morphine after lamellar keratectomy. Vet Ophthalmol. 2012;15(3):153-63.
  • 27
    Hirai Y, Migita K, Honda S, Ueki Y, Yamasaki S, Urayama S, et al. Effects of nitric oxide on matrix metalloproteinase-2 production by rheumatoid synovial cells. Life Sci. 2001;68(8):913-20.
  • 28
    Frears ER, Zhang Z, Blake DR, O'connell JP, Winyard PG. Inactivation of tissue inhibitor of metalloproteinase-1 by peroxynitrite. FEBS Lett. 1996;381(1-2):21-4.

Publication Dates

  • Publication in this collection
    Mar-Apr 2015

History

  • Received
    24 Sept 2014
  • Accepted
    01 Dec 2014
Conselho Brasileiro de Oftalmologia Rua Casa do Ator, 1117 - cj.21, 04546-004 São Paulo SP Brazil, Tel: 55 11 - 3266-4000, Fax: 55 11- 3171-0953 - São Paulo - SP - Brazil
E-mail: abo@cbo.com.br