The Department of Eye Histochemistry and Pharmacology is engaged in eye reseach, particularly of the anterior eye segment. The main research activities are aimed at the in situ and extra situm detection of enzymes as markers of various (patho)physiological states. Great attention is devoted to the use of various drugs (e.g., inhibitors of destructive proteases or scavengers of reactive oxygen species) with the aim of preventing (or at least diminishing) poor healing of ocular injuries or diseases that pose the danger of vision loss.
Current projects address the following:
- The healing of eye injuries
- Eye aging and premature eye aging, oxidative stress to the eye and antioxidant eye protection; hypoxia-reoxygenation injury of the cornea related to prolonged contact lens wear
- Environmental noxes and their influence on eye damage
- Polymers in ophthalmology (and dermatology)

      Plasminogen activation is an important mechanism of extracellular proteolysis; its primary role is to degrade extracellular matrices, including components of the intersticial matrix and basement membranes as well as fibrin clots. There are two plasminogen activators, urokinase-type (u-PA) and tissue-type (t-PA), both synthetized as single-chain enzymes, sc-uPA and sc-tPA. While sc-tPA has some activity as such, sc-uPA needs to be activated for full activity. A series of enzymes (including kallikrein, plasmin, cathepsin B, leukocyte elastase) can convert sc-uPA to active two-chain u-PA, which is potentially related to the mechanisms of the development of nonhealing epithelial corneal defects and/or stromal ulceration. Once active uPA is generated, conversion of plasminogen to plasmin quickly continues. Plasmin can activate latent matrix-degrading enzymes such as procollagenase. Furthermore, plasmin was found to be involved in the development of corneal and intraocular inflammation. In our Department, great attention has been devoted to the development of new histochemical and semiquantitative biochemical methods for the in situ and extra situm localization of various proteases and acid glycosidases (including plasmin, urokinase, leukocyte elastase, Cathepsin B, and acid beta-galactosidase.). For this reason, synthetic substrates with HFC (4 -trifluoromethylumbelliferyl) or AFC (7-amino-4-Trifluoromethyl Coumarine) leaving groups (purchased from Enzyme Systems Products, Dublin, CA, USA) have been employed. A knowledge of the level of enzyme activities in tissues, body fluids or secretions (e.g., tears, aqueous humor) is useful for the evaluation of early eye damage (e.g., associated with contact lens wear) or more profound disturbances, including ulcerative processes, and enables us to use specific inhibitors of destructive enzymes with the aim of improving the healing processes.

Fig1: On the right: Corneal stroma before ulceration. In the damaged stroma of a rabbit cornea burned with severe alkali (1N NaOH), beta-galactosidase activity is observed extracellularly. On the left: The activity of beta-galactosidase is increased in the superficial layers of a rabbit corneal epithelium irradiated with UVB rays.

      The conversion of molecular oxygen to its more reactive species (reactive oxygen species, ROS) has been suggested to be involved in aging processes (including the eye) and in the genesis of a variety of diseases. The pathological changes associated with aging processes are supposedly initiated by ROS-triggered unwanted reactions such as the oxidation of enzymatic and nonenzymatic soluble and structural proteins, membrane and cytosolic lipids, the degradation of nucleic acids and glycosaminoglycans, and the depletion of higher molecular weight antioxidants (enzymes: catalase, superoxide dismutase, glutathione peroxidase and reductase) as well as smaller molecular weight regulators (e.g., glutathione and ascorbate).

Fig.2: Distribution of xanthine oxidoreductase in the epithelium (on the left) and endothelium (on the right) of a human cornea (normal post-mortem human eye).

Fig. 3: An unhealed defect of the corneal epithelium reveals urokinase activity.

Fig 4: Upper portion: The urokinase activity in the endothelium of a rabbit cornea irradiated with UVB rays. Lower portion: The development of the retrocorneal membrane after an alkali burn of a rabbit cornea with 1N NaOH.

      The eye is directly exposed to chemical noxes that are very dangerous to the eye (mainly alkaline) and to various environmental noxes, such as ozone, radiation (including UV rays), cigarette smoke, nitric oxide and reactive oxygen species (ROS) generated by their influence. Under various conditions the anterior eye segment protective mechanisms are overwhelmed, resulting in eye damage. Under normal conditions antioxidants in the anterior eye segment are balanced with the formation of ROS at a level at which these compounds can play their physiological roles without any toxic effects. However, various noxes, e.g. UVB rays, evoke a decrease in antioxidant enzymes, whereas oxidases-generated ROS (namely xanthine oxidase) remain at high activity or are even increased. This might be dangerous to the eye due to insufficient ROS cleavage.

      UV absorbers covalently bound in hydrophilic contact or intracameral lenses, as well as water soluble polymers as drug carriers (releasing a drug - e.g. a specific enzyme inhibitor at the site of enzyme activation), have been developed in the Institute of Macromolecular Chemistry, Academy of Sciences of Czech Republic. The efficacy of UV absorbers for eye protection against UV rays and water soluble polymers as drug carriers for the improvement of wound healing have been investigated in our Department. Our results show that these materials are effective and useful for clinical practice.

Fig. 5: The demonstration of leukocyte elastase in inflammatory cells present in the stroma of a rabbit cornea.