Effects of Acetylcysteine on Rabbit Conjunctivol and Corneol Surfaces

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1 Investigative Ophthalmology & Visual Science, Vol. 32, No. 11, October 1991 Copyright Association for Research in Vision and Ophthalmology Effects of Acetylcysteine on Rabbit Conjunctivol and Corneol Surfaces A Scanning Electron Microscopy Study Florence Thermes, Sylvain Molon-Noblor, and Jeffrey Grove Conjunctiva! and cornea! epithelial surfaces of normal rabbit eyes with their associated mucus were studied by scanning electron microscopy before and after treatment with the mucolytic agent N-acetylcysteine (AC). Four groups received topically one 5-/il drop of either (Group A).1 M AC, (Group B).1 M AC every 5 min for 1 hr, (Group C).1 M AC every 5 min for 2 hr, or (Group D) three drops of 2% AC over 15 min. The effects of the instillation of AC on mucus removal and cellular lesions increased in the order (A) < (B) < (C) < (D). Treatment A had no effect on cornea and conjunctiva. Treatment B cleaned away mucosal debris without alteration of either conjunctival or corneal epithelium. Treatment C had a similar effect on the mucus but was associated with focal necrosis, and treatment D produced widespread necrosis, desquamation of epithelial cells, and inflammation. Invest Ophthalmol Vis Sci 32: ,1991 N-Acetylcysteine (AC) wasfirstused by Webb 1 as a mucolytic agent for the liquefaction of tenacious bronchial secretions. Its efficiency was confirmed in another study that found AC to be the least irritating and the most stable among several sulfhydryl agents. 2 Because of its mucolytic and anticollagenolytic properties, AC has been used in ophthalmology to treat corneal diseases, 34 such askeratoconjunctivitis sicca, 3 filamentous keratitis, 5 and corneal catarrh, and alkali-burned corneas. 6 ' 7 Corneal toxicity of AC was observed after intracorneal injection, 8 and evaluation of its effects on the cornea and mucus was made using light microscopy 91 and histochemistry." However, a detailed morphologic examination of the effect of AC on the ocular surfaces was not done to our knowledge. Although different treatments with AC were described, 9 ' the effective dose without undue toxicity has not been established. We therefore examined rabbit conjunctival mucosa and cornea using scanning electron microscopy (SEM) after topical application of various solutions of AC currently recommended. From the Centre de Recherche, Merck Sharp & Dohme-Chibret, Riom, France. Submitted for publication: February 15, 1991; accepted June 21, Reprint requests: Florence Thermes, MSc, Centre de Recherche, Merck Sharp & Dohme-Chibret, Riom, France. Materials and Methods Materials We purchased AC from Sigma (La Verpilliere, France), and solutions were prepared fresh on the day of the experiment. The ph was adjusted to 8 with.1 M NaOH. 14 All other chemicals were analytic grade and obtained commercially. New Zealand rabbits were supplied by Charles River (France). They were housed individually with a 12-hr light-12-hr dark cycle and had free access to food and water. Our investigations conformed to the ARVO Resolution on the Use of Animals in Research. AC Instillation The rabbits were placed in wooden restraining boxes to minimize movement. Unilaterally, we instilled 5-AII drops of AC into the left conjunctival sac, and the lower lid was brought gently up to meet the upper. The contralateral eye was not treated and served as the control. Three rabbits were used for each of the treatment groups reported in Table 1. Macroscopic examination of corneal and conjunctival surfaces was done on all rabbits before sampling. The animals were killed by rapid injection into the marginal ear vein of a lethal dose of sodium pentobarbital. Biopsy specimens were shaved off the lower pal Downloaded From: on 4/5/218

2 No. 1 ACETYLCY5TEINE EFFECTS ON CONJUNCTIVA AND CORNEA / Thermes er ol 2959 Table 1. Protocol design for topical application of N-acetylcysteine in rabbit left eyes (untreated right eyes were used as controls) "ftf? A Treatment groups B C D AC concentration Volume instilled No. of instillations Intervals between instillations Sacrifice.1 M 5 jil 1 3 min.1 M 5 jul 12 5 min 1 hr.1 M 5 nl 24 5 min 2hr 1.23 M (2%) 5^1 3 5 min 15 min fp-- :- pebral conjunctiva (5X5 mm) and off the cornea (3 X 3 mm). During the processing of samples, care was taken not to touch the specimens at any time before the SEM examination. Fig. 1. Scanning electron micrograph of untreated conjunctival surface showing typical hexagonal epithelial cells and randomly interspersed intercellular crypt openings (arrows), XI. *? -4 4 SEM Biopsy specimens were fixed by immersion in a 2.5% glutaraldehyde solution in.1 M sodium cacodylate buffer, ph 7.4, at 4 C for 2 hr. They were rinsed rapidly in.2 M cacodylate buffer and then fixed in a 1% osmium tetroxide solution in.1 M cacodylate buffer for 1 hr at room temperature. The specimens were dehydrated in a series of graded alcohol solutions, immersed in amyl acetate, and subsequently dried in liquid carbon dioxide using a critical-point drying apparatus (CPD1; Balzers Union, Liechtenstein). They were mounted with silver-conducting paint on aluminum stubs and coated with gold and palladium in a sputtering apparatus (SCD3; Balzers) under a residual argon atmosphere. We used a SEM (JSM T2; Jeol, Tokyo, Japan) under an accelerating voltage of 25 kev. To evaluate the effect of AC in quantitatively, each biopsy specimen was graded on a scale of -3 during the SEM examination. A value of 3 was ascribed to typical control eyes and when no trace of mucus was found. Two individuals did the assessments; the number of points then were totaled, and a mean was obtained of the "mucus index" for each treatment group. Results No visible signs of intolerance to AC were observed, except with the 2% AC solution; the latter induced marked ocular irritation after instillation. Macroscopic examination of the ocular surfaces showed a redness of the conjunctiva in treated eyes from this group. All other changes described hereafter were observed by SEM. Conjunctiva! Surface Under scanning illumination, the palpebral conjunctiva of untreated eyes had a wavy surface formed by the juxtaposition of irregular polygonal cells with marginal folds delineating the intercellular contacts (Fig. 1). At high magnification, the luminal membranes of epithelial cells were covered uniformly with dense microvilli. Goblet cell apertures, some of them extruding mucus droplets, were interspersed randomly between epithelial cells (Fig. 2). Mucus secretions present on the conjunctival surfaces were represented by disseminated droplets and a thin homogeneous mucus layer entrapping the microvilli of epithelial cells (Fig. 3). Fig. 2. Goblet cell discharging a mucus droplet onto the conjunctival surface, X5. Downloaded From: on 4/5/218

3 296 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / Ocrober 1991 Vol. 32 Fig. 3. Mucus secretions visible on the conjunctival surface consist of disseminated droplets (asterisk) and a mucus layer covering and sticking microvilli of epithelial cells (arrows), X2. Fig. 5. After a 2-hr application of.1 M acetylcysteine, the conjunctival surface shows focal cell degeneration and presence of inflammatory cells (arrows), X1. Local administration of one 5-^1 drop of.1 M AC (Group A) did not change either the appearance of epithelium or mucus secretions at 3 min postinstillation. After repeated application of this concentration every 5 min for 1 hr (Group B), the mucus layer covering the microvilli disappeared from extensive areas, resulting in a clean conjunctival surface (Fig. 4). However, isolated mucus secretions represented by droplets and strands were still present in some areas. In this group, AC effects were limited to the mucus layer, and the morphology of epithelial ceils was preserved. Repeated application of.1 M AC over 2 hr (Group C) had similar effects on the mucus as those of Group B. Conjunctival surfaces were clear of the mucus layer, and only a few mucus droplets were present. In Group C, all treated conjunctivas showed focal cellular alterations, consisting of degeneration and desquamation of epithelial cells, and the presence of inflammatory cells (Fig. 5). Instillation of 2% AC every 5 min over 15 min (Group D) also resulted in the disappearance of the mucus layer and focal degeneration and inflammation of the epithelium. Changes observed in this group affected all treated eyes and were more severe and extensive than in other groups. For a qualitative appraisal of the effects of AC on the conjunctival mucus, histograms were constructed to quantify (1) the mucus layer covering the conjunctival surface (Fig. 6A) and (2) the secretion of mucus (Fig. 6B), represented by mucus droplets. This evaluation showed good reproducibility of the control values, which were always consistently between , with the exception of one group. Thus, the histogram (Fig. 6A) showed that, compared with the control eyes, a dose-related reduction of the mucus layer occurred in the AC-treated eyes. At the same time, mucus secretion was almost unchanged, and the index only decreased to 2 with 2% AC (Fig. 6B). Corneal Surface Fig. 4. Repeated application of.1 M acetylcysteine for 1 hr results in a clear appearance of the conjunctival surface. Epithelial cells are not covered with a mucus layer and raicrovilli are clearly visible, X35OO. The surface of the cornea of untreated eyes observed by SEM consisted of characteristic flat polygonal cells arranged in a mosaic pattern, in which light, dark, and intermediate cells were distinguishable (Fig. 7). High magnification revealed numerous microvilli covering the cell surfaces (Fig. 8). Table 2 summarizes the incidence and visual signs of toxicity of the cornea after instillation of the various AC solu- Downloaded From: on 4/5/218

No. 11 ACETYLCYSTEINE EFFECTS ON CONJUNCTIVA AND CORNEA / Thermes er ol 2961 A. MUCUS LAYER COVERING THE SURFACE 2- t 3.3- control 3.' treated 2.5-1.5-1 A B D N.

4 No. 11 ACETYLCYSTEINE EFFECTS ON CONJUNCTIVA AND CORNEA / Thermes er ol 2961 A. MUCUS LAYER COVERING THE SURFACE 2- t 3.3- control 3.' treated A B D N.Acetylcystaine pretreatment % ->- B- SECRETION OF MUCUS N.Acetylcysteine pretreatrnent control treeted Fig. 6. (A) The "mucus index" of control and treated eyes calculated for the mucus layer covering the conjunctival surface (see conjunctival mucus for details). Three animals were used in each group. (B) The "mucus index" of control and treated eyes calculated for the secretion of mucus granules (see conjunctival mucus for details). Three animals were used in each group. tions; no changes were observed.5 hr after a single application of.1 M AC (Group A). After repeated application of.1 M AC for 1 hr (Group B) and 2 hr (Group C), changes in epithelial surfaces were observed in one and two animals, respectively. These changes consisted mainly of local loss of microvilli on the cell surfaces (Fig. 9), together with cell degeneration and desquamation of necrotic cells (Fig. 1). After repeated application of 2% AC for 15 min (Group D), similar changes were observed, but more animals were affected. Fig. 7. The corneal surface observed in untreated eyes consists of the juxtaposition of flat polygonal cells in which light (LC), dark (DC), and intermediate cells (IC) are distinguishable, X5. therefore the instrument of choice for the study of the disposition of mucus on the ocular surfaces. The heterogeneous mucus layer, entrapping microvilli and mucus droplets randomly extruded from the conjunctival surface, is seen clearly in our SEM photographs. This mucus layer 16 is spread over the globe during blinking and stabilizes the precorneal tear film between successive blinks Our results demonstrate the mucolytic effect of AC on this conjunctival mucus layer. A dose-related effect of AC on the mucus layer was found, as illustrated by the histogram (Fig. 6 A). Nevertheless, despite the fact that the ocular surfaces are clean after repeated AC treatment, mucus droplets continue to be secreted to protect the eye (Fig, 6B) against AC. Discussion The morphology of ocular structures has been investigated widely using light and transmission electron microscopy. More recently, SEM 1315 was used to study the normal and pathologic ultrastructure of ocular surfaces to evaluate the local tolerance of ophthalmic preparations. This technique has several advantages. It has a larger magnification range and better depth of focus and can provide detailed three-dimensional views of morphologic structures. The SEM was Fig. 8. High magnification shows microvilli covering the cell surface of the corneal epithelium, X75. Downloaded From: on 4/5/218

5 2962 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / Ocrober 1991 Vol. 32 Table 2. Incidence and visual signs of toxicity of the cornea after instillation of acetylcysteine solutions Modifications observed in corneal epithelium /. Number of animals with modification B,n = 3 C, n = j» A «= 3 Loss of microvilli Cell degeneration Cell desquamation Treatment group, t Number of animals. The mechanism of action of AC on mucus was described earlier. 2 In this previous study, the presence of free sulfhydryl groups were found necessary to reduce the viscosity of mucoprotein solutions, and increasing the concentration of AC decreased the viscosity of the mucus. A lower mucus viscosity therefore would lead to a more rapid removal from precorneal and preconjunctival areas. Our observations by SEM agree with these earlier findings 2 and provide further evidence for the effect of increasing AC concentration on mucus removal. All four AC applications we administered affected the mucus to a greater or lesser extent. It would appear that, because of the continuous formation of mucus, regular 5-min instillations of AC are necessary to maintain a clean surface. The 1-hr treatment with.1 M AC (Treatment B) was effective in removing mucus without any evident toxicity. However, toxic effects of AC on ocular structures were seen after 2 hr (Treatment C) or with three drops of 2% AC (Treatment D). The 1.23 M AC solution (Treatment Fig. 9. Repeated application of.1 M acetylcysteine for 2 hr results in a partial or total loss of microvilli on the surfaces (asterisk) together with deformation of plasma membranes (arrows), XI. Fig. 1. Necrosis and desquamation of comeal epithelial cells are observed after repeated application of acetylcysteine, X5. D) was hyperosmolar, and the.1 M AC solution (Treatments A-C) was almost isotonic. The toxicity may be caused by the hyperosmolarity of the solution, but others found no severe damage with a 6% NaCl hyperosmolar solution. 1 This is the first time that SEM has been used to demonstrate the superficial necrosis and cellular infiltration in the conjunctiva and cornea of the rabbit after instillation of 2% AC to our knowledge. Previous reports on the toxicity of AC did not agree with our findings. No adverse effects were found in studies on the rate of reepithelialization of the rabbit cornea with 1% and 2% AC after a superficial epithelial ulcer. 9 By contrast, others observed that an intracorneal injection of 2% AC, which is available commercially, rapidly caused "in vivo" disintegration of the corneal stroma. 8 Our results support the latter findings of toxicity of AC at this concentration. In conclusion, SEM was used to show a dose-related effect of AC on the maintenance of the mucus layer. A.1 M solution of AC was adequate to dissolve the mucus deposited on the conjunctival and corneal surfaces by blinking. It was necessary to administer several applications to ensure a clean ocular surface. A satisfactory treatment, producing little observable toxicity, was 5-fil drops at 5-min intervals for 1 hr. Longer periods, or treatment with 2% AC, produced marked toxic lesions in rabbits. Because this species is known to be extremely sensitive to irritation, 19 the relevance of these results to humans must be established with clinical data Key words: acetylcysteine, conjunctiva, cornea, mucus, scanning electron microscopy.< -fc,.. Downloaded From: on 4/5/218

No. 11 ACETYLCYSTEINE EFFECTS ON CONJUNCTIVA AND CORNEA / Thermes er ol 2963 Acknowledgment The authors thank Madame Marjorie Cremont for preparing the manuscript. References 1.

6 No. 11 ACETYLCYSTEINE EFFECTS ON CONJUNCTIVA AND CORNEA / Thermes er ol 2963 Acknowledgment The authors thank Madame Marjorie Cremont for preparing the manuscript. References 1. Webb WR: Clinical evaluation of a new mucolytic agent acetylcysteine. J Thorac Cardiovasc Surg 44:33, Sheffner AL: The reduction in vitro in viscosity of mucoprotein solutions by a new mucolytic agent, N-acetyl-L-cysteine. Ann N Y Acad Sci 16:299, Absolon MJ and Brown CA: Acetylcysteine in keratoconjunctivitis sicca. Br J Ophthalmol 52:31, Slansky HH, Berman MB, Dohlman CH, and Rose J: Cysteine and acetylcysteine in the prevention of corneal ulcerations. Ann Ophthalmol 2:488, Haut J, Labrune P, Ullern M, and Chermet M: New trial treatment of dry eye with acetylcysteine ophthalmic solution. Bull Soc Ophtalmol Fr 77:165, Brown S and Weller CA: Collagenase inhibitors in prevention of ulcers alkali-burned cornea. Arch Ophthalmol 83:352, Brown S, Tragakis M, and Pearce D: Treatment of the alkaliburned cornea. Am J Ophthalmol 74:316, Obenberger J and Cejkova J: Corneal damage following intracorneal injection of N-acetyl-L-cysteine. Graefes Arch Clin Exp Ophthalmol 185:171, Petroutsos G, Guimaraes R, Giraud JP, Renard G, and Pouliquen Y: Effect of acetylcysteine (Mucomyst) on epithelial wound healing. Ophthalmic Res 14:241, Sugar A and Waltman SR: Corneal toxicity of collagenase inhibitors. Invest Ophthalmol 12:779, Nichols BA, Chiappino L, and Dawson CR: Demonstration of the mucous layer of the tear film by electron microscopy. Invest Ophthalmol Vis Sci 26:464, Calabria G, Gandolfo E, Rolando M, and Zingirian M: N- aceylcysteine hypertonic solution in corneal diseases. In The Cornea in Health and Disease: Vlth Congress of European Society of Ophthalmology, Trevor-Roper PD, editor. London, Academic Press, 1981, pp Menna F, Antonucci R, Ippolito S, Maronne V, and Matrisciano F: Emploi de collyres a la cysteine et a l'acetylcysteine dans les brulures chimiques de la cornee. Bull Mem Soc Ophtalmol Fr 94:425, Ranga Rao KV, and Buri P: A novel in situ method to test polymers and coated microparticles for bioadhesion. Int J Pharm 52:265, Renard G, Montanez-Mendoza M, Savoldelli M, and Pouliquen Y: Etude en microscopie a balayage de l'epithelium corneen. J Fr Ophtalmol 6:697, Greiner JV, Korb DR, Covington HI, Peace DG, and Allansmith MR: Human ocular mucus. Arch Ophthalmol 1:1614, Holly FJ and Lemp MA: Wettability and wetting of corneal epithelium. Exp Eye Res 11:239, Ziment I. Acetylcysteine: A drug that is much more than a mucokinetic. Biomed Pharmacother 42:513, Duprat P and Conquet P: Predictive value of ocular irritation tests. Adv Vet Sci Comp Med 31:173, Downloaded From: on 4/5/218

Effect of Acetylcysteine on Experimental Corneal Wounds in Dogs

Original Paper Ophthalmic Res 2003;35:319 323 DOI: 10.1159/000074070 Received: July 8, 2002 Accepted after revision: May 27, 2003 Effect of Acetylcysteine on Experimental Corneal Wounds in Dogs S.J. Aldavood