Histochemistry(1992) 97: 69-76

Histochemistry © Springer-Verlag 1992

Histochemical changes in the rabbit cornea and plasmin activity in the tear fluid during contact lens wear. Favourable influence of protease inhibitors (aprotinin, PC5, elastatinal) J. (~ejkovfi 1, Z. Lojda 2, J. Vacik 3, G.A. Digenis 4, and S. Drop~ova 1 1 Institute of Experimental Medicine,CzechoslovakAcademyof Sciences, Legerova61, 120 00 Praha 2, CSFR 2 Laboratory of Histochemistry,Facultyof Medicine, Charles University,Studni6kova2, 120 00 Praha 2, CSFR 3 Institute of MacromolecularChemistry,CzechoslovakAcademyof Sciences, Heyrovsk6honfim~sti2, 160 00 Praha 6, CSFR 4 Collegeof Pharmacy,Universityof Kentucky,Lexington,KY 40536, USA Accepted August 17, 1991

Summary. Plasmin activity in the tear fluid of the rabbit eye was examined during the wearing of soft contact lenses (SCL) and compared with the occurrence of corneal disturbances assessed in cryostat sections. Plasmin activity was determined with a semiquantitative method using dry punches of filter paper previously soaked in 0.1 M Tris-HC1 buffer solution containing mmol/! D-Val-Leu-Lys-FCA (trifluoromethylaminocoumarine), pH 7.2. Punches were applied to the corneal surface for 5 s (tear collection) and incubated in wet chamber. The time of appearance of the bright yellow fluorescence in UV light was recorded and taken as a measure of plasmin activity. For calibration punches soaked in solutions containing plasmin in various concentrations, and processed in the same manner were used. Changes in the cornea were examined histochemically using methods of choice for acid glycosidases, proteases, dehydrogenases, and Na +-K +-ATPase. SCL with high and low water content were worn in rabbits in 1, 2, 4, 7, 14, 21 and 28 days. Decreased activity of Na+-K+-ATPase, GGT, and SDH in the corneal endothelium and epithelium were not accompanied by detectable plasmin activity in the tear fluid. Pronounced damage of the corneal epithelium (increased activities of acid glycosidases, acid proteases, LDH, markedly decreased activity of SDH) was accompanied by low concentration of plasmin (0.4-1.0 gg/ml) in the tear fluid. Middle activity of plasmin (1.0 2.0 pg/ ml) was detectable when PMNs were present in the corneal stroma. High plasmin activity (2.0 3.0 ~tg/ml) correlated with corneal ulceration and vascularization. The occurrence of both - plasmin activity and corneal disturbances was highly dependent on the water content of SCL (which goes parallel with oxygen permeability), duration of SCL wear, mechanical stress, and bacterial contamination. Mechanical irritation is considered to be the main factor leading to the appearance of plasmin activity in the tear fluid. The local application of aprotinin which inhibits plasmin and some other serine proOfft)rint requests to: J. Cejkov~

teases, enables us to prolong the harmless wear of SCLH (approximately one week). The combination of aprotinin with leukocyte elastase inhibitors (elastatinal and particularly PC5), prevents ulceration of the cornea and inhibits corneal vascularization after SCLL wear. Vascularization of the cornea does not occur if protease inhibitors are combined with flurbiprofen, an anti-inflammatory drug of cyclooxygenase pathway of arachidonic acid. Protease inhibitors also improved the course of bacterial keratitis.

Introduction Contact lens wear in humans may cause disturbances of the corneal epithelium (Hamano et al. 1983 ; Hayashi et al. 1985; Holden et al. 1986; Madigan et al. 1987), acute and chronic keratitis (Zantos and Holden 1978; Josephson and Coffery 1979), ulceration (Adams et al. 1983; Weissman et al. 1984) and vascularization of the cornea (Dixon and Lawaczeck 1963; Dixon 1967; Weinberg 1977; Nirankari etal. 1983). Hypoxia (Masters 1988), mechanical stress (Thoft and Friend 1975; Kilp et al. 1985), bacterial contamination (Ruben et al. 1976; Kersley et al. 1977), and other factors (Lohman 1986) were considered to play a role in these processes. In rabbit cornea, destructive processes accompanying contact lens wear culminate in ulceration and vascularization of the cornea connected with the infiltration by enzymatically active polymorphonuclear leukocytes (PMNs) (Cejkovfi et al. 1988). PMNs appear in the corneal stroma after various wounding: deepithelization (Srinivasan and Kulknari 1980), incision wounds (Weimar 1957; Matsuda and Smelser 1973), alkali burns (Kenyon etal. 1979; Chayakul and Reim 1982; Cejkovfi and Lojda 1988), wearing of contact lenses particularly of those with low oxygen permeability (Cejkovfi et al. 1988). Hydrolytic enzymes leaked from PMNs cause epithelial damage and

70 s t r o m a l m a t r i x d e g r a d a t i o n ( K e n y o n et al. 1979; P a h litzsch a n d S i n h a 1985; Cejkovfi et al. 1989). In the p a t h o g e n e s i s o f p e r s i s t e n t o r r e c u r r e n t epithelial defects a n d c o r n e a l u l c e r a t i o n after a l k a l i b u r n , a n essential role was a s c r i b e d to the d e g r a d a t i o n o f f i b r o nectin b y p l a s m i n ( B e r m a n e t a l . 1988; B a r l a t i e t a l . 1990). P l a s m i n , a serine p r o t e a s e , is g e n e r a t e d f r o m plasm i n o g e n b y active p l a s m i n o g e n a c t i v a t o r ( W a n g et al. 1985). Because p l a m i n o g e n a c t i v a t o r m a y be secreted b y c o r n e a l epithelial cells, k e r a t o c y t e s a n d P M N s , c o n v e r s i o n o f p l a s m i n o g e n to p l a s m i n p r o c e e d s q u i c k l y a n d leads to a n i n c r e a s e d p l a s m i n c o n t e n t in the t e a r fluid. In h u m a n s S a l o n e n et al. (1988) a n d Tervo a n d Setten (1989) r e p o r t e d o n a c o r r e l a t i o n b e t w e e n p l a s m i n activity in the tear fluid a n d s o m e c o r n e a l lesions. These lesions c o u l d be p r e v e n t e d b y local a p p l i c a t i o n o f a p r o t i n in, a n i n h i b i t o r o f p l a s m i n ( S a l o n e n et al. 1987). In the alkali b u r n e d a n t e r i o r eye s e g m e n t o f the r a b b i t severe c o r n e a l lesions were p r e v e n t e d using high c o n c e n t r a t i o n o f a p r o t i n i n d r o p p e d i m m e d i a t e l y after the i n j u r y o n the eye surface (Cejkovfi et al. 1988). I n these experim e n t s a p r o t i n i n also i n h i b i t e d the i n v a s i o n o f P M N s i n t o the c o r n e a l s t r o m a , a n d a c t i v a t i o n o f their enzymes. It a c c e l e r a t e d the r e e p i t h e l i z a t i o n o f the c o r n e a . A f t e r c o n t a c t lens w e a r in h u m a n s Tervo et al. (1989) a n d Setten et al. (1990) f o u n d a n i n c r e a s e d p l a s m i n a c t i v i t y in the t e a r fluid a s s o c i a t e d w i t h a h i g h e r degree o f c o r n e a l v a s c u l a r i z a t i o n a n d l i m b a l injection. H o w e v e r , d e t a i l e d i n f o r m a t i o n h a s n o t been available. F u r t h e r m o r e , the effect o f the a p p l i c a t i o n o f p r o t e a s e i n h i b i t o r s o n the c o u r s e o f c o r n e a l lesions c a u s e d b y c o n t a c t lens w e a r has n o t been studied. I n c o n t r a d i s t i n c t i o n to h u m a n s , studies in e x p e r i m e n t a l a n i m a l s are a d v a n t a g e o u s d u e to the p o s s i b i l i t y o f a d e t a i l e d s t u d y o f the c o r n e a l d i s t u r bances. We d e c i d e d t h e r e f o r e to p e r f o r m such studies on r a b bits. O u r a t t e n t i o n was f o c u s e d o n several p o i n t s : 1) To define the c o n d i t i o n in w h i c h p l a s m i n a c t i v i t y occurs in the t e a r fluid d u r i n g c o n t a c t lens wear, a n d to c o m p a r e the level o f p l a s m i n a c t i v i t y w i t h c o r n e a l d i s t u r bances. 2) To e x a m i n e w h e t h e r c o r n e a l a l t e r a t i o n s m a y be i n f l u e n c e d b y local a p p l i c a t i o n o f a p r o t i n i n (an i n h i b i t o r o f p l a s m i n ) , e l a s t a t i n a l a n d PC5 ( i n h i b i t o r s o f l e u k o cyte elastase), a n d f l u r b i p r o f e n (an a n t i - i n f l a m m a t o r y d r u g ) f o u n d p r e v i o u s l y to i n h i b i t v a s c u l a r i z a t i o n o f the c o r n e a ( D u f f i n et al. 1982).

Materials and methods Experiments were carried out on adult Chinchilla rabbits (3.54.0 kg). The eye,lids were slightly distended and sterile soft contact lenses (SCL) were applied to the cornea of both eyes. SCL were prepared in the Institute of Macromolecular Chemistry of the Czechoslovak Academy of Sciences to fit the rabbit cornea (12.5 mm curvature radius, 0.2 mm central as well as peripheral thickness, 15 mm diameter). The water content of SCL was 37 (L) and 65% (H) respectively. In one group of animals SCLL, in the other group SCLH were used. Lenses were worn continuously 1, 2, 4, 7, 14, 21 or 28 days. After these time intervals lenses were removed and plasmin activity was measured in the tear fluid on the corneal surface. In other groups of animals with SCLL and SCLH the following drug solutions were dropped on the eye surface for

28 days: Aprotinin (Trasylol, Bayer, Leverkusen, FRG, 60 UI/ml of saline), elastatinal (Sigma, St. Louis, Mo., USA, 50 gg/ml of saline), PC5 (tetrapeptide carbamate, a novel elastase inhibitor, kind gift from Professor Digenis, Lexington, Kentucky, USA, 50 gg/ml of 1.5% propylene glycol in saline), Flurbiprofen (Sigma, 1 mg/ml of saline). Aprotinin, elastatinal, PC5, and Flurbiprofen were applied either alone or combined by mixing the same amount of individual drug solutions. The drug solutions were dropped 4 x daily. In control experiments saline was used. Plasmin activity in the tear fluid (on the corneal surface) was measured after the removal of contact lenses in the same time intervals of SCL as in experiments without inhibitor application. Afterwards the animals were killed in thiopental narcosis and their corneas examined (see below).

Assessment of plasmin activity Punches of Whatman 1 filter paper (5 mm of diameter) were soaked in the solution of D-Val-Leu-Lys-FCA (trifluoromethylaminocoumarine, Enzymes System Products, Livermore, Calif., USA), and dried. The substrate solution was prepared as follows: 1 mg of substrate was dissolved in 4 drops of N,N-dimethylformamide and 1 ml of 0.1 M Tris-HC1buffer, pH 7.2 added. Human plasmin solutions containing 0.2-0.5 Ixg of plasmin (Sigma) dissolved in 0.1 M Tris-HC1 buffer were prepared; 20 gg of every sample was dropped onto punches which were then incubated (in wet chamber) at 37° C in a thermostat and observed in 2-min intervals in UV light (UVGL-25, manufactured by CVP, Inc., San Gabriel, Calif., USA). The time of appearance of bright yellow fluorescence was recorded. Plasmin concentration > 3.0 ~tg/ml was detectable after 15 min incubation. For this concentration the designation "very high activity" is used. The fluorescence of samples with "high plasmin activity" (2.0-3.0 gg/ml) appeared after 30 min of incubation. Samples with "middle activity" (1.0-2.0 ~tg/ml) furnished fluorescence after i h incubation. "Low activity" (0.4-1.0 lag/ml) was detectable after 1.5 h incubation. In punches soaked in samples containing a lower concentration of plasmin than 0.4 gg/ml and incubated 2 h no fluorescence was detected (plasmin negativity). For practical purpose it is advantageous to incubate punches for 2 h. After this time interval differences among the given groups of plasmin activity (very high, high, middle, and low can be clearly distinguished). Plasmin activity in the tear fluid was assessed in the following way: Punches with substrate were applied onto the corneal surface and let 5 s to soak. Afterwards they were removed and incubated in a wet chamber. Fluorescence in UV light was observed in 15 min intervals, and compared with that of callibrated punches.

Histochemical and morphological examination After killing the animals the eyes were immediately enucleated and the corneas cut out. One portion of corneas was treated with silver impregnation technique according to Mc Govern (1955). After stripping of epithelium and endothelium the preparations were mounted in polystyrene. The other portion of corneas was fixed in 4% formaldehyde containing 1% CaC12 for 2 h and embedded in paraffin, 10 gm sections cut perpendicularly to the corneal surface were stained with Harris haematoxylin and eosin. The third group of corneas was quenched in light petroleum, chilled with acetone-dry ice mixture, and cut in a cryostat. Sections (12 gm) cut parallel or perpendicular to the corneal surface were transferred to non-precooled slides and used for the detection of glycosaminoglycans and enzymes. Sections adherent to semipermeable membranes were used for the demonstration of acid glycosidases. For the examination of the glycosaminoglycans, sections were fixed in cold ethanol (5 min), and stained with 1% aqueous solution of Alcian blue containing various concentration of MgC12 (0.2 Mr_ 1.2 M MgC12) (Cejkovfi et al. 1973), and also with 1% aqueous solution of Alcian blue (1%) in 3% acetic acid, pH 2.5. For the demonstration of alkaline phosphatase, 7-glutamyl transferase,

71 aminopeptidase M, and dipeptidyl peptidase IV sections were fixed with cold chloroform-acetone mixture (1 : 1) for 2 rain (Lojda et al. 1979; Lojda 1984; Cejkovfi and Lojda 1986). Alkaline phosphatase was demonstrated using azo-coupling method with naphthol-ASMX-phosphate (Calbiochem, La Jolla, Calif., USA) and Fast Blue BB Salt (Michrome, Gurr, Poole, UK) or Variamine blue RT Salt (Dajac, Chicago, Ill., USA). Aminopeptidase M (APM) was assessed with Ala-4-methoxy-2-naphthylamine (MNA, Bachem, Bubendorf, Switzerland) and Fast Blue B (FBB, Michrome, Gurr), Dipeptidyl peptidase IV (DPP IV) with Gly-Pro-MNA (Bachem) and FBB, ?,-glutamyl transferase (GGT) with 7-Glu-MNA (Bachem), Gly-Gly (Bachem) and FBB (Lojda et al. 1979; Cejkovfi and Lojda 1986). Na+-K+-dependent adenosintriphosphatase (ATPase) was examined by the method using ATP-Tris or Baz÷ salt according to Cejkovfi and Lojda (1978). Dehydrogenase (succinate dehydrogenase, SDH, lactate dehydrogenase, LDH) were detected in unfixed cryostat sections using methods recommended by Lojda et al. (1979) with nitro BT (Lachema, Brno, CSFR) as electron acceptor. Sections on semipermeable membranes were employed for the demonstration of acid phosphatase (naphthol AS-BI-phosphate, Calbiochem), and hexazonium-p-rosaniline prepared from acridinfree p-rosaniline (Merck, Darmstadt, FRG), fl-glucuronidase (naphthol AS-BI-fl-D-glucuronide and hexazonium p-rosaniline), and N-acetyl-fl-D-glucosaminidase,Calbiochem (naphthol-AS-BIfl-D-glucosaminide and hexazonium p-rosaniline). Acid fl-galactosidase was demonstrated with the indigogennic method of Lojda using 4-C1-5-Br-3-indolyl-fl-D-galactoside(Cyclo Chemicals, Los Angeles, Calif., USA) (Lojda et al. 1979). The last portion of the corneas was fixed in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.2 (4° C, 12 h), washed in 0.1 M phosphate buffer, pH 7.2, quenched and cut in a cryostat; 12 gm sections were transferred to albuminized slides and allowed to thaw and dry. Fixed sections were used for the demonstration of dispeptidyl peptidase I (DPP I) (Gly-Arg-MNA, Bachem, and nitrosalicylic aldehyde, NSA, Merck), DPP II (Lys-Pro-MNA, FBB), (Lojda 1985; Cejkov/t and Lojda 1986).

Results

Low plasmin activity ( < 0.4 lag/ml) in the tear fluid occurs after 4-7 days of continuous wear of SCLL and after 7-14 days of SCLH. In the corneal epithelium there is observed a loss of activities of Na+-K+-dependent ATPase, G G T , SDH, decreased activity of alkaline phosphatase in superficial cells, highly increased activities of acid phosphatase, acid glycosidases and of lysosoreal proteases DPP I and II (Figs. 1, 2). Using the Mc Govern method (stripped corneal epithelium), disturbances in the pattern of corneal cells are found. In cases in which SCL wear causes a greater mechanical irritation (Fig. 10), enzymatic changes and plasmin activity and the tear fluid appears earlier. In the corneal stroma, keratocytes displayed decreased activities of alkaline phosphatase and CGT, and in the corneal endothelium a loss of activities of N a + - K + - A T P a s e and G G T takes place. Using the Mc Govern method polymegatism in the corneal endothelium is seen. Low to middle activity of plasmin (0.4-1.5 gg/ml) in the tear fluid occurs after 7-14 days of continuous wear of SCLL, and after 14-21 days of SCLH. In addition to the described alterations of the corneal epithelium, increased activities of acid glycosidases and some proteases (DPP I, II, IV) are found in keratocytes beneath the epithelium (Figs. 3, 4). Using the Mc Govern method

small and large cells occur in corneal epithelium due to the pressure influences (Fig. 11). In cases with pronounced epithelial defects are very well detected using the Mc Govern method (Fig. 12). Some inflammatory ceils are present in the anterior stroma and in the distored epithelium. In those cases plasmin activity is higher (2.0 gg/ml). Middle to high plasmin activity (1.5-2.5 gg/ml) in the tear fluid is found after 14-28 day wear of SCLL. In the corneal epithelium high activities of acid glycosidases and lysosomal proteases are present. In the corneal stroma, inflammatory cells with elevated activities of lysosomal hydrolases appear (Figs. 5, 6). These enzymes release and cause local destructions with the loss of stainability of glycosaminoglycans with Alcian blue in 3% acetic acid (pH 2.5) culminating in ulceration or vascularization of the cornea (Figs. 7, 8). These changes coincide with middle (1.0-2.0 gg/ml) to high activity of plasrain (2.(~3.0 gg/ml) in the tear fluid. In the corneal endothelium pronounced polymegatism is visible. Some endothelial cells are lost. In the cases of SCLH middle to high plasmin activity is detectable only in those cases when bacteria are involved or pronounced irritation of the corneal epithelium takes place.

The effect of enzyme inhibitors (aprotinin, elastatinal, PC5), and anti-inflammatory drug (flurbiprofen ) Aprotinin. When aprotinin is applied from the first day of continuous SCLH wear (by imbibition of SCLH or by dropping its solutions on the eye surface covered with SCLH 4 x daily), the epithelial disturbances occur later (approximately a week). No plasmin activity can be detected. In later stages the amount of PMNs is highly decreased (Fig. 14). The infection is inhibited both in the case of SCLH and SCLL wear. The extent of the corneal vascularization is not changed.

Elastatinal and PC5. The effect of both drugs is very similar: PC5 seems to be a more potent inhibitor. After the local application of these drugs, a decreased amount of P M N s is detected and corneal vascularization is partially prevented. The inhibitory effect on P M N invasion is more pronounced when leukocyte elastase inhibitors are applied together with aprotinin (Fig. 15). Elastatinal and PC5 do not inhibit plasmin activity in the tear fluid. However, after local application of leukocyte elastase inhibitors, plasmin activity appears later and its levels are lower in comparison to cases without treatment. The inhibitory effect on the infection is highly pronounced.

Flurbiprofen. This drug applied locally by dropping on the eye surface markedly inhibits corneal vascularization. The amount of PMNs is lower.

The combination of protease inhibitors with flurbiprofen, and the combination of aprotinin, elastatinal (or PC5) and flurbiprofen prevents severe corneal lesions even after long-term wear of SCLL. Is must be stressed that prevention of ulceration and vascularization of the cor-

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Figs. 1-8. Corneal alterations after continuous SCLL wear

Fig. 1. DPP I activity in corneal epithelium after 2 days of SCLL wear is normal, x 160 Fig. 2. DPP I activity is increased in a thinned epithelial layer after 7 days of SCLL wear. x 160 Fig. 3. After 10 days of SCLL wear the activity of fl-glucuronidase is increased in the corneal epithelium and also in some keratocytes beneath the epithelium, x 160 Fig. 4. After 14 days of SCLL wear the activity of fi-glucuronidase in keratocytes beneath the epithelium is very high. x 160

n e a is successful p a r t i c u l a r l y in cases w i t h o u t m e c h a n i c a l irritation.

The combination of aprotinin, and elastatinal (or PC5) is also a d v a n t a g e o u s for the h e a l i n g o f local d e s t r u c t i v e processes in the c o r n e a after r e m o v a l o f SCL.

Discussion O u r e x p e r i m e n t s w i t h S C L w e a r in r a b b i t s s h o w t h a t p l a s m i n c o n c e n t r a t i o n in the tear fluid a n d c o r n e a l dis-

Fig. 5. A great number of inflammatory cells with high activity of/~-glucuronidase is present in the anterior stroma after 21 day wear ofSCLL, x 160 Fig. 6. DPP I activity after 21 day SCLL wear is very high in the epithelium and inflammatory cells beneth the epithelium, x 160 Figs. 7 and 8. The stainability of glycosaminoglycans in the corneal stroma (Alcian blue, pH 2.5, counterstaining with Nuclear fast red) after 28 days of SCLL wear. In Fig. 7 the arrowpoints in the direction from the posterior part of the stroma towards the middle part of the stroma where vessels are present. In the anterior part of the stroma (arrowpoints from the middle to the upper stroma) numerous PMNs can be seen. (Fig. 8). x 240

t u r b a n c e s are highly d e p e n d e n t o n the w a t e r c o n t e n t o f S C L (which goes p a r a l l e l w i t h the o x y g e n p e r m e a b i l i ty ( H a m a n o a n d K a u f m a n 1987)), lasting o f c o n t a c t lenses, m e c h a n i c a l stress a n d the presence o f infection. H o w e v e r , these f a c t o r s d o n o t influence the p l a s m i n activity in the tear fluid to the s a m e extent. F i r s t c h a n g e s due to w e a r i n g o f S C L w i t h u n s u f f i c i e n t o x y g e n p e r m e a b i l i t y ( H a m a n o a n d K a u f m a n 1987) are f o u n d in the c o r n e a l e n d o t h e l i u m f o l l o w e d b y the altera t i o n s o f the e p i t h e l i u m : d e c r e a s e d activities o f N a ÷K + - d e p e n d e n t A T P a s e , G G T , a n d S D H . These c h a n g e s are n o t a c c o m p a n i e d b y d e t e c t a b l e p l a s m i n activity in

73

Figs. %12. Results of the Mc Govern's method in the stripped corneal epithelium

Fig. 11. After 21 days of SCLH wear superficial epithelial cells become irregular. Small and large cells can be seen. x 160

Fig. 9. After 4 days of SCLH wear superficial layer of corneal epithelial cells is normal, x 160 Fig. 10. After 4 days of SCLL wear superficial epithelial cells are destroyed in places where mechanical irritation occurs. The a r r o w points to the defect, x 160

Fig. 12. After 21 days of SCLL wear greater destruction of superficial epithelial cells appears in the area of more pronounced mechanical influences. The a r r o w points to the destroyed area. × 160

the tear fluid. Plasmin activity appears later when the d a m a g e o f corneal epithelium is m o r e p r o n o u n c e d (highly increased activities o f acid glycosidases, acid p h o s p h a tase, e.g. D P P I, II, o f L D H , and m a r k e d l y decreased activity o f S D H . Plasmin activity in the tear fluid and corneal disturbances a p p e a r after short-term wear o f S C L L a n d p r o l o n g e d wearing o f S C L H . I f mechanical irritation is remarkable (defects o f corneal epithelium are shown in Figs. 10, 12), plasmin activity in the tear

fluid together with corneal disturbances occur immediately, independent o f the water c o n t e n t o f SCL. We suppose that mechanical irritation is the m a i n factor evoking the appearance o f plasmin activity in the tear fluid and that h y p o x i a is less important. It is questionable, whether h y p o x i a alone can be a cause o f plasmin occurrence in the tear fluid. A c c o r d i n g to M a n g u m et al. (1987) h y p o x i a m a y n o t exert a direct release o f plasminogen activator. It influences the course

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Figs. 13-15. Influence of protease inhibitors on the pattern of the cornea during the continuous wear of SCLL Fig. 13. fl-Galactosidase activity in the anterior portion of the cornea after 21 days of SCLL (placebo-treated corneas). Great number of highly active inflammatory cells is present, x 180

Fig. 14. fl-Galactosidase activity in corneas after 21 days of SCLL wear which were locally treated with aprotinin (4 x daily). The amount of inflammatory cells is highly reduced, x 180 Fig. 15. fl-Galactosidase activity in corneas treated with aprotinin combined with elastatinal. Only some inflammatory cells are present in the anterior stroma, x 180

of inflammation which is more serious if microbes are present. P M N s arrive at the damaged area of the cornea (Srinivasan and Kulknari 1980) by migration from perilimbal vessels (Robb and Kuwabara 1962). P M N s are highly involved in destructive corneal processes (Kenyon et al. 1979; Chayakul and Reim 1982; Cejkovfi and Lojda 1988). They are present both in the corneal stroma and in the tear fluid (Srinivasan and Kulknari 1980). The number of P M N s is influenced by several chemotactic factors. Microbial products have been found to be chemotactic for P M N s (Badenoch and Coster 1988). Plasmin generates chemotactic fragments for P M N s from complement (O'Flaherty and Ward 1979). Prostaglandins and other metabolites of arachidonic acid may also be involved in the mediation of P M N chemotaxis (Srinivasan and Kulknari 1980). In this connection it is worth mentioning that reduced oxygen levels can lead to an increased production of prostaglandins (Lands 1979). However, it is possible that hypoxia may evoke the appearance of plasmin in the tear fluid indirectly via enzymes released from PMNs. These enzymes damage corneal cells which may secrete plasminogen activator, converting immediately plasminogen to plasmin. On the other hand corneal cells can be damaged directly (by mechanical insults) without mediating influence of PMNs. However, P M N s contribute to the lesions of corneal cells in later stages similarly as in the case in which the cornea is damaged by alkali or other chemical burns. Damaged corneal cells secrete plasminogen activator after chemical injury (Berman et al. 1983; Wang 1985;

Chan 1986). After deepithelization of the cornea plasmin activity occurs in the tear fluid in higher levels and decreases with the reepithelization of the cornea (our unpublished results). In the case of SCL wear, plasmin activity quickly appears in the tear fluid in cases of pronounced mechanical irritation by SCL. In some cases vascularization of the cornea takes place. The vascularization of the cornea may also be a sequel of mechanical irritation and action of enzymes of plasminogen activator/plasmin system. Berman et al. (1985) found that angiogenesis in areas of tissue damage is mediated by the secretion of plasminogen activator. According to our experiments, plasmin is involved in the origin and development of some corneal lesions during SCL wear. Plasmin degrades fibronectin (Vartio et al. 1981; Vaheri et al. 1985; Vaheri and Salonen 1988), stimulates the secretion of latent forms of proteases from various cell types and converts them to active forms (Wang et al. 1985; Cejkovfi et al. 1989). Furthermore, it contributes to the invasion of PMNs into the corneal stroma (Berman et al. 1983). Since plasmin and elastase proved to be harmful in corneal damage, the use of inhibitors of proteases seemed to be highly desirable. These inhibitors could block the damaging effect. Three protease inhibitors were used in our study: Aprotinin, an inhibitor of serine proteases plasmin, neutrophilic elastase, and some other proteases, elastatinal, and PC5 - inhibitors of neutrophilic elastase. Besides, flurbiprofen (an anti-inflammatory agent) was also tried. These drugs were applied separately and in combination.

75 A p r o t i n i n a p p l i e d d r o p w i s e o r using c o n t a c t lens s o a k e d in its solution, p r e v e n t e d the u l c e r a t i o n a n d v a s c u l a r i z a tion o f the c o r n e a f r o m h a p p e n i n g . M i l d e r c h a n g e s in the cases o f S C L H w e a r t o o k p l a c e at least one w e e k later. T h e use o f l e u k o c y t e elastase i n h i b i t o r s (elastatinal, PC5) was also successful. L e u k o c y t e elastase, a serine p r o t e a s e , a t t a c t s a wide v a r i e t y o f s t r u c t u r a l l y i m p o r t a n t p r o t e i n s a n d g l y c o p r o t e i n s . T h e y i n c l u d e elastin, fibronectin, p r o t e o g l y c a n , c o l l a g e n fibres (Digenis et al. 1986; B o d e et al. 1989; Skiles et al. 1990). E l a s t a t i n a l , a n d p a r t i c u l a r l y PC5, i n h i b i t i n v a d i n g P M N s a n d the v a s c u l a r i z a t i o n o f the c o r n e a . L e u k o c y t e elastase i n h i b i t o r s d o n o t i n h i b i t p l a s m i n activity. H o w e v e r , after their local a p p l i c a t i o n , p l a s m i n c o n c e n t r a t i o n in the t e a r fluid occurs later a n d reaches l o w e r levels in c o m p a r i s o n to unt r e a t e d cases. A c o m b i n a t i o n o f a p r o t i n i n a n d PC5 p o tentiates the i n h i b i t o r y effect o f i n d i v i d u a l drugs. F l u r b i p r o f e n p r e v e n t s the v a s c u l a r i z a t i o n o f the c o r n e a a n d lowers the n u m b e r o f P M N s . T h e c o m b i n a t i o n o f serine p r o t e a s e i n h i b i t o r s w i t h f l u r b i p r o f e n enables us to use l o w e r c o n c e n t r a t i o n o f f l u r b i p r o f e n . Such a d r u g c o m b i n a t i o n is useful p a r t i c u l a r l y for the h e a l i n g o f c o r n e a l ulcers after r e m o v i n g o f S C L (cases o f l o n g - t e r m w e a r of SCLL without dropping of inhibitors). It c a n be c o n c l u d e d t h a t p r o t e a s e i n h i b i t o r a p r o t i n i n enables us to p r o l o n g significantly S C L H w e a r in r a b bits. A p r o t i n i n w i t h PC5 (elastatinal) p r e v e n t s u l c e r a t i o n a n d v a s c u l a r i z a t i o n o f the c o r n e a after S C L L wear, especially w h e n c o m b i n e d with f l u r b i p r o f e n . E a r l y findings w i t h S C L w e a r e r s s h o w t h a t a similar c o m b i n a t i o n of protease inhibitors and anti-inflammatory drug could be efficient in the p r e v e n t i o n a n d t r e a t m e n t o f c o r n e a l lesions in h u m a n s .

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