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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)


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-HCl buffer solution containing mmol/l 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 μg/ml) in the tear fluid. Middle activity of plasmin (1.0–2.0 μg/ml) was detectable when PMNs were present in the corneal stroma. High plasmin activity (2.0–3.0 μg/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 proteases, enables us to prolong the harmless wear of SCLH (approximately one week). The combination of aprotin-in 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.

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  1. Adams CP, Cohen E Jr, Laibson PR, Galantine P, Arentsen JJ (1983) Corneal ulcers in patients with cosmetic extended-wear contact lenses. Am J Ophthalmol 96:705–716

  2. Badenoch PR, Coster DJ (1988) Antibiotics and corticosteroids: functions and interaction in ocular disease. In: Cavanagh HD (ed) The cornea: Transactions of the World Congress on the Cornea III. Raven Press, New York, pp 475–483

  3. Barlati S, Marchina E, Quaranta CA, Vigasio F, Semerano F (1989) Analysis of fibronectin, plasminogen activators and plasminogen in the tear fluid as markers of corneal damage and repair. Exp Eye Res 51:1–9

  4. Berman M, Manseau E, Law M, Aiken D (1983) Ulceration is correlated with the degradation of fibrin and fibronectin at the corneal surface. Invest Ophthalmol Vis Sci 24:1358–1376

  5. Berman M, Kenyon K, Hayashi K, L'Hernault N (1988) The pathogenesis of epithelial defects and stromal ulceration. In: Cavanagh HD (ed) The cornea: Transactions of the World Congress on the Cornea III. Raven Press, New York, pp 35–43

  6. Bode W, Meyer JR, Powers JC (1989) Human leukocyte elastase and porcine pancreatic elastase: X-ray crystal structures, mechanism, substrate specificity, and mechanism-based inhibitors. Biochemistry 28:1951–1963

  7. Čejková J, Bolková A, Lojda Z (1973) A study of acid mucopolysaccharides in cold microtome sections of normal and experimentally hydrated bovine corneas. Histochemie 36:167–172

  8. Čejková J, Lojda Z (1978) Occurrence and importance of Na+-K+-dependent adenosintriphosphatase in the cornea. (In Czech). Čs Oftalmologie 6:401–404

  9. Čejková J, Lojda Z (1986) Histochemistry of some proteases in the normal rabbit, pig and ox cornea. Histochemistry 84:67–71

  10. Čejková J, Lojda Z (1988) Histochemistry of proteases in alkali burned rabbit cornea. Dependence on the concentration of alkali and the mode of its application. Biol Zentralbl 107:201–206

  11. Čejková J, Lojda Z, Brůnová B, Vacík J, Michálek J (1988) Disturbances in the rabbit cornea after short-term and long-term wear of hydrogel contact lenses. Histochemistry 89:91–97

  12. Čejková J, Lojda Z, Salonen E-M, Vaheri A (1989) Histochemical study of alkali-burned rabbit anterior eye segment in which severe lesions were prevented by aprotinin treatment. Histochemistry 92:441–448

  13. Chan KY (1986) Chemical injury to an in vitro ocular system: differential release of plasminogen activator. Curr Eye Res 5:357–362

  14. Chayakul V, Reim M (1982) Enzymatic activity of β-N-acetylglucosaminidase in the alkali-burned cornea. Graefes Arch Clin Exp Ophthalmol 218:149–152

  15. Digenis GA, Agha BJ, Tsuji K, Kato M, Shinogi M (1986) Peptidyl carbamates incorporating amino acid isosteres as novel elastase inhibitors. J Med Chem 29:1468–1476

  16. Dixon JM (1967) Corneal vascularization due to corneal contact lenses: the clinical picture. Trans Am Ophthalmol 65:333–340

  17. Dixon JM, Lawaczeck E (1963) Corneal vascularization due to contact lenses. Arch Ophthalmol 69:72–75

  18. Duffin RM, Weissman BA, Glasser DB, Pettit TH (1982) Flurbiprofen in the treatment of corneal neovascularization induced by contact lenses. Am J Ophthalmol 93:607–614

  19. McGovern VJ (1955) Reactions to injury of vascular endothelium with special reference to the problem of thrombosis. J Pathol Bacteriol 69:283–285

  20. Hamano H, Hori M, Hamano T, Kuwabe H, Mitsunaga S, Hamano T (1983) Effects of contact lens wear on mitosis of corneal epithelium and lactate content in aqueous humor of rabbit. Jpn J Ophthalmol 27:451–458

  21. Hamano H, Kaufman HE (1987) Physiology of the cornea and effect of contact lens wear. In: Hamano H, Kaufman HE (eds) The physiology of the cornea and contact lens application. Churchill Livingstone, New York Edinburgh London, pp 13–35

  22. Hayashi I, Tada T, Kishimoto H (1985) H-thymidine incorporation into corneal epithelia of rabbits under continuous wear of oxygen-permeable hard contact lenses. J Jap Contact Lens Soc 27:153–158

  23. Holden BA, Sweeney DF, Vannas A, Nilsson KT, Efron N (1986) Effects of long-term extended contact lens wear on the human cornea. Invest Ophthalmol Vis Sci 26:1489–1502

  24. Josephson JE, Caffery BE (1979) Infiltrative keratitis in hydrogel lens wearers. Int Contact Lens Clin 6:223–241

  25. Kenyon KR, Berman M, Rose J, Gage J (1979) Prevention of stromal ulceration in the alkali-burned rabbit cornea by glued-on contact lens. Evidence for the role of polymorphonuclear leukocytes in collagen degradation. Invest Ophthalmol Vis Sci 18:570–587

  26. Kersley HJ, Kerr C, Pierse D (1977) Hydrophilic lenses for “continuous wear” in aphakia: definitive fitting and the problems that occur. Br J Ophthalmol 61:38–42

  27. Kilp H (1985) Metabolites and enzymes in the corneal epithelium after extended contact lens wear. Curr Eye Res 4:738–739

  28. Lands WEM (1979) The biosynthesis and metabolism of prostaglandins. Ann Rev Physiol 41:633–652

  29. Lohman LE (1986) Corneal epithelial response to contact lens wear. Clao J 12:153–156

  30. Lojda Z, Gossrau R, Schiebler TH (1979) Enzyme histochemistry. A laboratory manual. Springer, Berlin Heidelberg New York

  31. Lojda Z (1984) Die Histochemie der Proteasen. Acta Histochem 30 [Suppl]:9–29

  32. Lojda Z (1985) The importance of protease histochemistry in pathology. Histochem J 17:1063–1089

  33. Madigan MC, Holden BA, Kwok LS (1987) Extended wear of contact lenses can promise corneal epithelial adhesion. Curr Eye Res 6:1257–1260

  34. Mangum M, Venable RH, Boatwright JD, Cocke TB (1987) Hypoxia: a stimulus for tissue plasminogen activator release in humans? Aviat Space Environ Med 58:1093–1096

  35. Masters BR (1988) Effects of contact lenses on the oxygen concentration and epithelial mitochondrial redox state of rabbit cornea measured noninvasively with an optically sectioning redox fluorometer microscope. In: Canavagh HD (ed) The cornea. Transactions of the World Congress on the Cornea III. Raven Press, New York, pp 281–286

  36. Matsuda H, Smelser GK (1973) Electron microscopy of corneal wound healing. Exp Eye Res 16:424–427

  37. Nirankari VS, Karesh J, Lakhanpal V, Richards RD (1983) Deep stromal vascularization associated with cosmetic, daily-wear contact lenses. Arch Ophthalmol 101:46–47

  38. O'Flaherty JT, Ward PA (1979) Chemotactic factors and the neutrophil. Semin Hematol 16:163–166

  39. Pahlitzsch T, Sinha P (1985) The alkali-burned cornea: electron microscopical, enzyme histochemical, and biochemical observations. Graefes Arch Clin Exp Ophthalmol 223:278–286

  40. Robb RM, Kuwabara T (1962) Corneal wound healing. I. The movement of polymorphonuclear leukocytes into corneal wounds. Am J Ophthalmol 68:636–642

  41. Ruben M, Brown N, Lobascher D, Chaston J, Morris J (1976) Clinical manifestation secondary to contact lens wear. Br J Ophthalmol 60:529–531

  42. Salonen E-M, Tervo T, Törmä E, Tarkkanen A, Vaheri A (1987) Plasmin in tear fluid of patients with corneal ulcers: basis for new therapy. Acta Ophthalmol 65:3–12

  43. Salonen E-M, Lauharanta J, Sim P-S, Stephens R, Vaheri A (1988) Rapid appearance of plasmin in tear fluid after ocular allergen exposure. Clin Exp Immunol 73:146–148

  44. Van Setten GB, Tervo T, Anderson R, Perheentupa J, Tarkkanen A (1990) Plasmin and epidermal growth factor in the tear fluid of contact-lens wearers: effect of wearing different types of contact lenses. Ophthalmic Res 22:233–240

  45. Skiles JW, Fuchs V, Chow G, Skoog M (1990) Inhibition of human leukocyte elastase by N-substituted tripeptide trifluoromethyl ketones. Res Commun Chem Pathol Pharmacol 68:365–373

  46. Srinivasan BD, Kulknari PS (1980) The role of arachidonic acid metabolites in the mediation of the polymorphonuclear leukocyte response following corneal injury. Invest Ophthalmol Vis Sci 19:1087–1093

  47. Tervo T, Setten GB van (1989) Aprotinin for inhibition of plasmin on the ocular surface: principles and clinical observations. In: Beuerman RW, Crosson CE, Kaufman HE (eds) Healing processes of the cornea. Porfolio, The Woodlands, pp 151–163

  48. Tervo T, van Setten GB, Anderson R, Salonen E-M, Vaheri A, Immonen I, Tarkkanen A (1989) Contact lens wear is associated with the appearance of plasmin in the tear fluid: preliminary results. Graefes Arch Clin Exp Ophthalmol 227:42–44

  49. Thoft RA, Friend J (1975) Biochemical aspects of contact lens wear. Am J Ophthalmol 80:139–145

  50. Vaheri A, Salonen E-M, Vartio T (1985) Fibronectin in formation and degradation of the pericellular matrix. In: Evered D, Whelan J (eds) Fibrosis. Ciba Foundation Symposium 111. Pitman, London, pp 111–126

  51. Vaheri A, Salonen E-M (1986) Fibronectin and regulation of proteolysis in cancer and tissue destruction. Proc Finn Dent Soc 84:13–18

  52. Vartion T, Seppä H, Vaheri A (1981) Susceptibility of soluble and matrix fibronectins to degradation by tissue proteinases, mast cells chymase and cathepsin G. J Biol Chem 356:471–477

  53. Wang HM, Berman M, Law M (1985) Latent and active plasminogen activator in corneal ulceration. Invest Ophthalmol Vis Sci 26:511–524

  54. Weimar V (1957) Polymorphonuclear invasion of wounded corneas. J Exp Med 105:141–152

  55. Weinberg RJ (1977) Deep corneal vascularization caused by aphakic soft contact lens wear. Am J Ophthalmol 83:121–122

  56. Weissman BA, Mondino BJ, Pettit TH, Hofbauer JD (1984) Corneal ulcers associated with extended-wear soft contact lenses. Am J Ophthalmol 97:476–481

  57. Zantos SG, Holden BA (1978) Ocular changes associated with continuous wear of contact lenses. Aust J Optom 61:418–426

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Čejková, J., Lojda, Z., Vacík, J. et al. 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). Histochemistry 97, 69–76 (1992).

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  • Plasmin
  • Keratitis
  • Flurbiprofen
  • Corneal Endothelium
  • Corneal Stroma