Diagnostic methods and treatment modalities of dry eye conditions.

One may view dry eye conditions as a group of diseases in which the ocular surface is adversely affected. Tear film instability invariably leads to some degree of cellular surface damage over the cornea and conjunctiva. In turn, ocular epitheliopathy may adversely affect tear film stability. The clinical presentation of the disease may not yield a clue as to its etiology. In recent years considerable progress was made both in the diagnosis and the treatment of the disease and promising studies are planned or are underway. The diagnostic techniques can be divided into four groups. The first is concerned with the clinical presentation. The second is concerned with the bulk properties of the aqueous tears including dynamic characteristics, composition, and colligative properties. The third is tear-film related and includes the film break-up time, evaporation rate, and lipid abnormality. The fourth is concerned with the ocular surface and includes vital staining, impression cytology, and surface microscopy. The most promising attempts are being made in the second group by attempting to elucidate the role of enzyme and enzyme activator activity and inhibitor contents as well as the tear protein profiles and correlating them with the specific disease states. The treatment modalities belong to three major groups aside from surgical intervention; the supplementation, preservation, and the stimulation of tears. The modern version of tear supplementation is expected to include the topical use of efficacious aqueous formulations that typically contain film stabilizing polymers, nutrients, and/or--in the future--biochemically active ingredients such as enzyme activators and inhibitors.

Tear plasminogen activators--indicators of epithelial cell destruction. The effect of scraping, n-heptanol debridement, and alkali burn of the cornea on the plasminogen activator activity of rabbit tears.

Plasminogen activator (uPA) activities were determined in the tears of rabbits following mechanical (scraping) or chemical (n-heptanol) debridement and alkali burn of the central part of the corneal epithelium. All three types of injury enhanced the plasminogen activator activities in the tears. The increase in uPA activity was highest in alkali burn, lowest for n-heptanol debridement. Scraping yielded an intermediate increase in uPA activity. The maximum value in activator activity was reached at 5 hours for mechanical injury and at 24 hours for chemical injuries. uPA activity values returned to the normal range by the time of re-epithelialization for mechanical scraping and 1-3 days following re-epithelialization for heptanol debridement and alkali burn. A trend was observed between uPA activity level and the size of the wound but the correlation was not pronounced (R = 0.538).

Determination of plasminogen activator activities in normal and pathological human tears. The significance of tear plasminogen activators in the inflammatory and traumatic lesions of the cornea and the conjunctiva.

Plasminogen activator activity in normal human tears was found to be 0.03 +/- 0.02 IU/ml with casein plate, and 0.06 +/- 0.04 IU/ml with a spectrophotometric method. Elevated levels of plasminogen activator activity (range 0.11-2.05 IU/ml) were detected in the tear fluid of patients suffering from various corneal and conjunctival diseases including corneal ulcers, superficial keratitis, persistent epithelial defects, recurrent erosions, bullous keratopathy, contact lens associated erosions, alkali burns of the cornea, Mooren's ulcer, conjunctival pemphigoid, acute keratoconus, and corneal melanoma. Plasminogen activator activity, determined in the absence of fibrin in tear samples collected by capillary tubes at low flow rates, is considered to be the result of the presence of urokinase-type plasminogen activator (uPA) deriving from the epithelial cells of the cornea and the conjunctiva. It is suggested that an increase in the level of uPA in tears plays an important role not only in ulceration (the formation and repair of epithelial and stromal defects), but also in the development and healing of a number of other inflammatory processes, infections, immunological processes, chemical burns, contact lens associated lesions; in the invasion of microorganisms and leukocytes, in edema formation, in neovascularization, and in the invasive growth of tumors in the cornea and the conjunctiva.

Comparison of lacrimation kinetics in dry eye patients and normals.

A 100 mm long and 5 mm wide filter paper strip enclosed in a transparent plastic sheath to prevent evaporation was used to determine the time dependence of wetting of the strip in the anesthetized eyes of dry eye patients and age-matched normals. Prior to the measurements, the paper strips were extracted by lipid solvents and marked at every millimeter length. Wetted length data were plotted against time and the resulting wetting curves were analyzed to obtain the time dependence of the tear secretion rate during lacrimation. Every eye studied exhibited an exponentially decaying tear secretion rate that could be characterized by three kinetic parameters per cycle: the initial and final tear secretion rates and the secretion decay coefficient. The eyes of the sicca patients showed a much simpler lacrimation pattern than did the controls: 60% of the dry eyes exhibited a one-cycle lacrimation pattern while only 5% of the normal group did so. The tear secretion kinetic parameters characterizing the lacrimation pattern reduced to one-cycle were compared. Both the dry eyes and the control eyes started to lacrimate at about the same high initial secretion rate. However, the lacrimation rate was found to decrease faster and to a lower final rate in dry eye patients as opposed to normal controls.

Physical chemistry of the normal and disordered tear film.

The preocular tear film is viewed as a thin, mostly aqueous film covering a hydrophilic solid and bordered by a meniscus which is also supported by the solid. The aqueous layer is coated by an even thinner lipid layer. Part of the solid boundary is mobile and capable of compressing the lipid layer rapidly thereby simulating blinking. By the use of such a model, the properties of the tear film system can be described and its behavior can be predicted by applying the principles of interface science to the model. Gravity has a negligible effect on the tear film. Hydraulic flow of tears can only occur in the meniscus. The rupture of the tear film is attributed to lipid contamination of the supporting solid making its surface hydrophobic. Blinking compresses the lipid layer and removes the lipid-contaminated, hydrophobic mucus from the epithelium-tear interface. The instability of the preocular tear film, which can be caused by several factors, appears to be characteristic of all dry eye states irrespective of aetiology.

Colloid osmotic pressure of artificial tears.

While the total osmolality of the aqueous tears and tear substitutes has received much attention in the past few years, the colloidal osmolality or the oncotic pressure (which includes the Donnan effect), has received practically no attention except for one single foreign publication. The colloidal osmolality of tears is twentyfold less than that of the corneal stroma, which in turn is less than 1% of the total osmolality of an isotonic solution, i.e. the magnitude of the colloidal osmolality is only a few hundreths of a per cent of total osmolality. This may be the reason why its role was thought to be unimportant by many researchers. Despite its relatively small magnitude when compared to total osmotic pressure, the oncotic pressure has been shown to play a major role in the maintenance of the water balance of bodily tissues and has been used as a diagnostic parameter in alveolar edema. The same principle has been used to formulate a collyrium, Dehydrex, or dextran-containing storage media for excised corneas such as the Kaufman-McCarey medium that have a colloidal osmolality at least equal to that of deturgescent corneal stroma. Such formulations are able to dehydrate corneal stroma even in the total absence of epithelium. Dehydrex has been shown to have a beneficial effect on damaged epithelium and is thought to be the drug of choice for the treatment of recurrent epithelial erosion when other treatment modalities have failed. In the present study, the total osmolality and the oncotic pressure of several artificial tear preparations presently marketed was determined and compared with the oncotic pressure of tears and the corneal stroma. We have found that the oncotic pressure of HypoTears is nearly sixty times higher than that of the leading artificial tear, thus it is comparable to the oncotic pressure of Dehydrex. We believe that the favorable patient acceptance of HypoTears is more likely due to this unusually high oncotic pressure than to its hypoosmolality. Such an artificial tear formulation should be effective in ameliorating microcystic epithelial edema and in increasing impaired epithelial adhesion to the underlying tissue in the cornea.

Timolol inhibits corneal epithelial wound healing in rabbits and monkeys.

To determine the effect of timolol maleate on corneal epithelial wound closure, 6.4-mm (diameter) epithelial defects were created chemically in five experiments. In rabbits, wound closure was not significantly altered by the administration of 0.5% Timoptic (a commercially prepared timolol maleate solution) every eight hours, but it was inhibited during the first 15 hours of healing when a 4% solution was applied every two hours. Monkeys received a 5% solution every 12 hours and were divided into treated and control groups. Two phases of wound healing were apparent: a rapid initial phase and a slower final phase. Wound closure was inhibited in treated eyes during the initial rapid phase. This experiment repeated on the same monkeys, but with the control and treated animals switched, yielded the same result. Another experiment, with the fellow eye of the monkey used as a control, showed no significant difference in wound closure.

Intracellular distribution and properties of cathepsin B in the rat retina and its inhibition by a cytosolic fraction.

The intracellular distribution and properties of cathepsin B in the rat retina are similar to those of cathepsin B in other mammalian tissues. The specific activity of cathepsin B was highest in the mitochondrial-lysosomal fraction [1650 +/- 450 pmole mg-1 protein hr-1 (mean +/- S.D.)]. Inhibition of cathepsin B activity by a cytosol fraction of retina is also described.

Decrease of lactoferrin concentration in the tears of myotonic muscular dystrophy patients.

Tears from myotonic muscular dystrophy (MMD) patients and normal controls were analyzed for their tear proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Lactoferrin comprised about 18% of the total tear protein in MMD patients as opposed to about 27% in normals. The albumin content relative to total protein in MMD tears was about 25% while the same value for normals is 13%. The lactoferrin/albumin ratio, was about 0.8 for MMD patients and about 2.1 for normals.

Changes in lysosomal enzyme activities of eye tissues in endotoxin-induced inflammation.

Lysosomal enzyme activities in rabbit ocular tissues were examined at various times after the intravitreal injection of endotoxin. Lysosomal enzyme activities in the aqueous, vitreous, and pigment epithelium-choroid were elevated 3 h after injection. It is deduced from the fluorescein-labeled dextran and SDS-polyacrylamide gel electrophoresis analysis of the aqueous, vitreous, and pigment epithelium-choroid from endotoxin injected rabbits that the elevation of lysosomal enzyme activities are probably due to a breakdown in blood-ocular barriers.

Biochemical and surface characteristics of human tear proteins.

Pooled human tear samples were subjected to biochemical and surface chemical analyses. Gel filtration chromatography yielded four fractions. The second fraction was the most surface active, closely followed by the first. By ionic exchange chromatography, the first fraction obtained by gel filtration chromatography could be further separated into three subfractions. The second of these subfractions was found to be the most surface active, even surpassing the combined surface activity of all the tear components. Altogether eleven protein components were identified in human tears by gradient polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS). Their molecular weights range from 16,000 to 400,000 daltons. Isoelectric focusing indicated that most of the tear proteins are negatively charged, whereas the rest are positively charged at physiological pH's.

Investigation of the accuracy of tear lysozyme determination by the Quantiplate method.

The accuracy of lysozyme concentration determination by the method of measuring lysis diameter in agarose gel slabs containing Micrococcus lysodeikticus bacteria uniformly suspended throughout the gel was determined for various methods of tear sample collection. The effects of storage of lysozyme solution samples at subzero temperatures for several days was also examined. It was found that a power rather than the suggested exponential dependence between the lysozyme concentration and lysis diameter provides the most accurate fit and thus should be used for interpolation. Storing samples frozen in glass capillaries lowered the lysozyme concentration in a predictable manner. When Weck-Cel sponges were used to collect the samples the lysozyme concentration was greatly diminished in a nonlinear manner because of internal adsorption. The relative loss (cause by adsorption) depended on the actual lysozyme concentration as well as on the sample volume/sponge weight ratio. Storing samples absorbed by such sponges in a frozen state further altered the results in an unpredictable way. The observation that smaller tear samples for a given sponge size yielded lower apparent values for lysozyme concentration casts doubt on findings that have reported lower lysozyme concentration in the tears of keratoconjunctivitis sicca patients, where either cellulose sponges or filter paper discs were used for tear collection.

Kinetics of capillary tear flow in the Schirmer strip.

The popular Schirmer test of tearing ability heretofore has never been analyzed kinetically and the interpretation of the results is usually based on implicit, often erroneous assumptions. We have analyzed the capillary flow of fluids in filter paper strips in vitro systems for the cases of unlimited supply and limited supply at constant rates, and in vivo using human subjects, who kinetically corresponded to limited supply having variable rates. The effects of evaporation, surface tension, viscosity, paper pore-size, and the wettability of the cellulose fibers in the paper were also studied. The results show that in the case of unlimited supply, the fluid absorption is kinetically identical to fluid uptake by a horizontal capillary. This implies that the paper strip can absorb fluids only at a certain maximum rate which decreases with time. At lower secretion rates, the rate of wetting length increase is linearly proportional to the secretion rate provided that evaporation is prevented. Evaporation increases with increased wetting length until a steady state is reached where the length of wetting remains constant in time. As long as the secretion rate remains below the maximum uptake rate of the paper strip, the paper and fluid characteristics have a negligible influence on the wetting rate thus the rate of wetting can provide quantitative information on the secretion rate. Wetting length versus time curves obtained in vivo can be best described mathematically by assuming that the initially high secretion rate exponentially decays to a lower, final value. From the data, the magnitude of the initial and final tear secretion rates as well as the secretion rate decay coefficient can be calculated. These physiologically relevant values quantitatively characterize the functioning of the lacrimal system and may have diagnostic value in detecting marginal lacrimal deficiencies and predicting poor contact lens tolerance.