Histol Histopathol (1998) 13: 553-564 Histology and 001: 10.14670/HH-13.553 Histopathology http://www.hh.um.es From Cell Biology to Tissue Engineering Invited Review Enzyme histochemistry of corneal wound healing J.Cejkova Department of Eye Histochemistry, Institute of Experimental Medicine, Academy of Sciences of Czech Republic, Prague, Czech Republic Summary. The usefulness o f e nzyme hi stoche mical considered. T he majo rit y of destruc ti ve processes are meth ods fo r the localizati on of enzymes as catalysts of resiste nt (or o nl y s li g htl y sens itive) to co nventio nal molecular interactions in the cell s and tissues of healing therapy (based on the use of anti-inflammatory drugs corn eal wounds is shown in rabbits. The current data on and/or antibioti cs) ( Burns e t a I. , 1989). Recently, the corneal wound healing in humans as well as in rabbits uncontroll ed actio n of pro teolyti c e nzymes, me tall o­ w ith p arti c ul a r refe re nce to serin e pro teases a re proteinases (Fini et aI. , 1992; Matsumoto et aI. , 1993), rev iewed. Some infla mma to ry m e di a to rs a re a lso enzymes of pericellular proteolysis (Tervo et aI. , 1992; di scussed. Pl asmin is a serine protease w hi ch is absent Vaheri et aI. , 1992) and reacti ve oxygen species (ROS) (or present onl y in very low concentration) in th e tear (i.e., hydrogen peroxide, singlet oxygen and oxygen-free flu id, and it s acti vity appears under various pathological radicals such as superoxide anion and hydroxyl radical) conditions in humans or fo ll owing experimental injuries (Fantone and Ward, 1982; Kehrer, 1993) generated from in rabbits. The role of increasing plasmin activity in the e nv iro nme ntal po lliuta nts or produced during inte r­ tear fl ui d in th e depending upon the severit y of corneal mediary metaboli sm by the action of various oxidases injury is evalu ated . Great atte nt io n is d evote d to has been supposed to pl ay th e key role in the path o­ cond itio ns leading to lo ng-lasting elevated levels of genesis. plasmin acti vity in the tear fluid correlated with corneal T he ha rmful actio n of proteases a nd ROS led to ul ceration. The diffe rences between the hi stochemical a tte mpts a t us ing va rio us pha rmaco logical age nts, pattern of untreated corneas or corneas treated with some including inhibitors of proteolyti c enzymes (Burns et aI. , serine protease inhibitors are shown, and the efficacy of 1989, Cejkova et a I. , 1989, 1992; Fini et aI. , 1992; Tervo th ese drugs is di scussed in light of present knowledge. et a I. , 1992; Tervo and Setten, 1992; Wentworth et aI. , 1992; Pate rson et aI., 1994) and scavengers o f ROS Key words: Cornea, Enzymes, Hi stochemistry, Wounds, (Nirankari et aI. , 1981; Cejkova and Lojda, 1994). The Healing efficacy of these drugs (in v itro o r in expe rime ntal animals) can be investi gated by biochemical methods, and much informati on can be accumulated using this Introduction approach. However, biochemical methods cannot furnish any info rmation about possible focal disturbances in Ul cerative and other destruc ti ve processes of the individual tissue layers; e.g., corn eal layers. On the other cornea are catastrophic complicati ons of ocul ar lesions hand, classical histological methods, w hich have usuall y that are difficult to manage and can result in the loss of been employed, cannot reveal metabolic alt erations of visio n. The pathogenesis of these processes (occuring as the corn ea. Thus, sens it ive his toche mical tests a re persiste nt corn eal epitheli al defects or deeper ul cers) required. For the detecti on of enzymes, histochemical or re ma in s large ly unsolved. In publis hed pa pe rs, a n immuno hi stochemi cal methods can be used (see Lojda et associati on with various direct or indirect insults to the aI. , 1991 fo r detail s). Immunohistochemical methods are eye, such as trauma (alkali burn or irradiation of the eye; very important ; however, because they detect enzymes e .g . with UV ra y s), immuno logica l de ra nge m e nt as proteins, an acti ve and inacti ve enzyme (proenzyme) (rhe umato id a rthritis), ocul a r in fecti on (bacte ri al o r cannot be distinguished and therefore a method fo r the fungal in fe ctions) and vitamin A deficie ncy (kerato­ localiza ti o n of e nzy me acti v ities is indis pe ns ible. malacia) has been described (Carubelli et aI. , 1990). The Enzyme activities can be revealed in situ by methods of role of various inflammatory mediators has also been enzyme hi stoche mistry (catalytic histochemistry). The de tecti on of acti ve e nzymes is of decisive import ance Offprin t requests to: Professor Jit ka Cej kova, MD , PhD., DSc, Head, because o nl y active enzymes can be involved in various Depart ment of Eye Histochemistry, Institute of Experimentat Medicine, processes, such as angiogenesis, chemotaxis, inflamma­ Videnska 1083, 14220 Prague 4, Czech Republic ti on and activati on of proenzymes. 554 Enzyme histochemistry of corneal wounds Enzyme histochemical methods (using enzymes as interventions are also discussed briefly. markers of metabolic disturbances) are useful for better understanding the mechanisms of corneal damage and Plasmin activity in the tear fluid and simple methods repair. The histochemical pattern of the experimentally­ of its detection injured rabbit cornea is shown in relationship with plasmin activity in the tear fluid. Plasmin is a serine Plasmin is a serine protease which appears after the protease which is absent (or present only in very low conversion of plasminogen by plasminogen activators concentration) in the normal eye. However, under (Berman et aI., 1983; Saksela, 1985; Wang et aI. , 1985; various pathological conditions in humans or following Vaheri et aI., 1992). Berman et al. (1988), Cejkova et al. experimental injuries in rabbits, plasmin activity appears (1989) and Hayashi et al. (1991) have shown that in the tear fluid. Elevated plasmin activity is thought to plasmin activity in the tear fluid increases in corneal be associated with corneal inflammation and the ulcerative processes. Plasmin activity in the tear fluid development of corneal ulcers or other destructive can be measured by various methods. However, two processes. On the other hand, some plasmin activity is simple methods exist which do not require expensive presumed to be necessary for the healing process. laboratory equipment (important for use in clinical Therefore, we compared plasmin activity in the tear fluid practice): the method of radial caseinolysis (Saksela, with the histochemical pattern of untreated corneas or 1981) as modified by Salonen et al. (1987); or a semi­ corneas treated with aprotinin (an inhibitor of plasmin quantitative fluorescence method which we developed and some other serine proteases) evaluating the healing (Cejkova et aI., 1989, 1992). This method uses filter as "efficient" or "inefficient". Cases of experimental paper punches soaked with 0-Val-Leu-Lys-7-amino-4- corneal injury, in which "efficient" corneal healing is Trifluoromethyl Coumarin (AFC) (Enzyme Systems achieved by leukocyte elastase inhibitors and ROS Products, Livermore, CA, USA) as a substrate for scavengers, are also mentioned. plasmin detection. Three ,ul of tears or other fluids are Histochemical methods used are summarized in dropped onto dry filter paper punches containing the Table 1 (see Lojda et aI., 1991 for details; Xanthine substrate. Incubation takes place at 37 DC, and the time oxidase activity was detected as recommended by of the first appearance of the yellow fluorescence Gossrau et aI., 1989, 1990). The localization and (evaluated under UV light) is recorded and compared activities of individual enzymes in the normal cornea with the appearance of yellow fluorescence in calibrated and during the healing process after experimental injury punches with a known activity (concentration) of ~ave been described by Cejkova et al. (1975a-c), plasmin (Sigma, Munich, Germany). In calibrated Cejkova and Lojda (1978, 1988a,b), Cejkova et al. punches, a plasm in concentration of >3.0 Ilg/ ml (1988, 1989, 1992, 1993), and Cejkova and Lojda (1994, was detectable after 15 min incubation. For this 1995a,b, 1996). concentration the designation "very high plas min In this review, results in experimental animals are activity" is used. The fluorescence of samples with "high compared with clinical findings. The current data on plasmin activity" (2.0-3.0 flglml) appeared after 30 min corneal healing and the theoretical basis for therapeutic incubation. Samples with "moderate activity" (1.0-2.0 Table 1. Enzyme histochemical methods used in our experiments ENZYME METHOD Na+·K+·dependent adenosintriphosphatase (Na+· K+·dependent ATPase) ATP + Pb2+ (inhibitor ouabain) y-glutamyl transpeptidase (GGD y-glutamyl-MNA + FBB Aminopeptidase M (APM) alanyl·MNA + FBB Dipeptidyl peptidase I (DPP I) gly-arg·MNA + NSA (or FBB) Dipeptidyl peptidase II (DPP II) Iys-pro-MNA or ala·pro·MNA + FBB (or NSA) Dipeptidyl peptidase IV (DPP IV) glycyl·prolyl·MNA + FBB Alkaline phosphatase naphthol AS-MX-phosphate + FBBB acid phosphatase naphthol AS·BI-phosphate + HPR (j-glucuronidase naphthol·AS-BI-(j·D·glucuronide + HPR or (j-galactosidase 4-CI-5-Br-3-indolyl-(j-D-galactoside Acetyl-(j-D-glucosaminidase naphthol AS-BI -N-acetyl-(j-D·glucuronide + HPR succinate dehydrogenase (SOH) succinate in water medium + NBT+menadione lactate dehydrogenase (LDH) lactate in medium + PVA + NBT catalase H20 2 + DAB (aminotriazole as inhibitor) xanthine oxidase hypoxanthine + Ce3 + + DAB + C02+ Abbreviations: for aminoacids according to "IUPAC-IUB Commission on Biochemical Nomenclature", Eur.