N.J. van Haeringen

Corneal epithelial permeability after instillation of ophthalmic solutions containing local anaesthetics and preservatives

The effect of the local anaesthetics oxybuprocaine (OBu) and tetracaine (Tetra) and the preservatives chlorhexidine (CH) and benzalkonium chloride (BAK) on corneal epithelial permeability was studied by fluorophotometry in normal human eyes. Five instillations of one drop ophthalmic solution of the compounds were administered to one eye at 2-minute intervals; a control solution was instilled into the fellow eye. The increase in corneal epithelial permeability, expressed as the permeability ratio between the treated and control eye, was not significant after instillation of the anaesthetics. The preservatives and the combination OBu + CH increased corneal epithelial permeability significantly (P less than 0.05). OBu + BAK and Tetra + BAK increased permeability to a far greater extent (P less than 0.005).

The origin of some enzymes in tear fluid, determined by comparative investigation with two collection methods

Abstract Comparative investigations of human tear fluid, collected in capillaries as well on filter paper strips, revealed that the enzymes lactate dehydrogenase (LDH), malate dehydrogenase (MDH), pyruvate kinase (PK), isocitrate dehydrogenase (ICDH), aldolase (ALD), glucose-6-phosphate dehydrogenase (G-6-PDH), sorbitol dehydrogenase (SDH), glutamate dehydrogenase (GLDH) and glutamate-oxalacetate transaminase (GOT), are not secreted by the lacrimal gland, but can be liberated into tear fluid after slight damage of the conjunctival and corneal epithelium. Hexokinase (HK) and glutamate-pyruvate transaminase (GPT) and the lysosomal enzymes α-galactosidase (α-gal) and β-hexosaminidase (β-hex), which are produced by the lacrimal gland, may be present in increased amounts after slight epithelial damage. A practical application of these findings is discussed.

Selective properties of the vitreous barrier

Experiments with several groups of chemically related substances showed that differences in penetration into the vitreous body of the rabbit must be attributed to the existence of a vitreous barrier. More than one mechanism is involved in the function of the vitreous barrier, one of which is selective in respect to lipoid solubility, while another mechanism responds to the electrical charge of molecules.

The peroxidase-thiocyanate-hydrogenperoxide system in tear fluid and saliva of different species

Abstract The occurrence of peroxidase (POD) and thiocyanate in saliva and tears of humans and different animals is not so common as would be expected for a generally acting POD-thiocyanate-hydrogenperoxide antibacterial system. Moreover there does not appear to exist any correlation between salivary or lacrimal POD activity and the thiocyanate concentration in these secretions. In fact only in human and guinea-pig saliva are the requirements fulfilled for an effective antibacterial action. The secretions in other species contain no demonstrable POD activity (rabbit) or too low a thiocyanate concentration (cat, guinea-pig, human, rat). The function of the POD-thiocyanate-hydrogenperoxide system as an alternative for the bacteriolytic enzyme lysozyme, which is lacking in the secretions of species other than human and some monkeys, must be excluded.

Cholesterol in human tear fluid.

Abstract Small and varying amounts of cholesterol and cholesterol esters were found in human tear fluid. Failure to establish a relationship between the cholesterol concentrations of plasma and tears is in accord with the concept that the origin of these lipids is not to be found in the lacrimal gland, but in the Meibomian gland. The “oily substance” sometimes found on contact lenses in patients with contact lens problems, is not related to cholesterol or to elevated cholesterol levels in tear fluid.

A comparison of the effects of non-steroidal compounds on the disruption of the blood-aquous barrier

The anti-inflammatory agents, diclofenac, flurbiprofen and indomethacin, were examined to determine their inhibitory effect on the breakdown of the blood-aqueous barrier in rabbit eyes, following paracentesis. In treated eyes, compared to the non-treated eyes, the increase in protein and fluorescein in the secondary aqueous was inhibited in a dose-dependent manner. Dose-response curves for the non-steroidal compounds were comparable and were in the range of 3×10−6–10−2 m . However, indomethacin did not completely inhibit the breakdown of the aqueous barrier. The observed effects were strongly dependent of the pH of the vehicle and partly correlated with lipid solubility of the agents.

Urea and the vitreous barrier of the eye.

Massive infusion of urea in rabbits demonstrated that the uneven distribution of urea over the eye has its origin in a different speed of penetration of urea into the tissues. No characteristic physico-chemical properties can be attributed to the vitreous barrier on the basis of these concentration gradient, since penetration of urea seems to be restricted by secondary factors.

Detection of aldehyde dehydrogenase activity in human corneal extracts

The major soluble protein in bovine corneal epithelial extracts is a 54 kD protein (BCP 54) which has recently been identified as the corneal aldehyde dehydrogenase. Although ALDH activity has been reported in human corneal extracts it was not yet clear whether this was identical with the 54 kD protein described in bovine corneas. To investigate this question, we studied human corneal extracts for the presence of ALDH using enzyme analysis, SDS-PAGE, native electrophoresis, isoelectric focusing and immunoblotting techniques. The corneal epithelium was the most active layer (8.46 +/- 1.9 IU/mg protein) followed by the stroma (2.83 +/- 0.56 IU/mg protein) and endothelium (0.06-3.6 IU/mg protein). When comparing substrate specificity between human and bovine corneal ALDH, using NADP as coenzyme, it was shown that the human enzyme preferred benzaldehyde whereas the bovine enzyme revealed the strongest enzymatic activity with hexanal. Human corneal ALDH was partly inhibited by disulfiram. Bovine and human cornea ALDH lost their enzymatic activity after heating at temperatures above 56 degrees C. Both human and bovine corneal extracts contained a prominent 54 kD protein which reacted with a rabbit anti BCP 54 antibody. Isoelectric focusing followed by enzyme staining in the gel revealed 5 human corneal isozyme species and 4 in bovine corneal extracts, migrating at a pH between 6.5 and 7.0. All isozymes could also be detected after immunoblotting with a rabbit anti BCP 54 antibody.

Comparison of enzymes of tears, lacrimal gland fluid and lacrimal gland tissue in the rat

A variety of enzymes were identified in rat tears and lacrimal gland fluid. The use of a tapered glass cannula in the opening of the excretory duct was found to be an useful method to collect samples of rat lacrimal gland fluid, i.e., the fluid directly originated from the main excretory duct of the lacrimal gland, uncontaminated by secretions from the Harderian gland and from desquamating conjunctival and corneal epithelial cells. Based upon comparison of the enzyme pattern in tears, lacrimal gland fluid and the lacrimal gland tissue, we concluded that the lacrimal gland is the primary tissue source for peroxidase (POD), amylase (AMY) and total protein in rat tears, while plasminogen activator and lactatedehydrogenase (LDH) may be present in tears primarily as secretion products from other ocular tissue sources. beta-hexosaminidase (beta-hex) is released from the lacrimal gland and from other ocular tissue sources as well.

Modulation of immunogenic keratitis in rabbits by topical administration of inhibitors of lipoxygenase and cyclooxygenase

Intrastromal injection with human serum albumin (HSA) in the rabbit cornea induced edema and a ring-shaped leukocyte infiltrate followed by neovascularization. The effect of topically administered lipoxygenase and cyclooxygenase inhibitors on this inflammatory keratitis was studied. The lipoxygenase inhibitors Bay 08276 and Rev 5901 and the cyclooxygenase inhibitor suprofen were given as 1% eye drops three times daily during the experiment. In eyes treated with lipoxygenase inhibitors leukocyte infiltration, neovascularization and edema formation decreased. In eyes treated with a cyclooxygenase inhibitor the period of neovascularization was slightly shortened and corneal edema decreased. No influence on leukocyte infiltration was seen.