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Background: Prolonged exposure to infrared (IR) radiation is associated with different types of damage to cornea and lens. The aim of our study was to investigate the effect of acute and chronic exposure to IR radiation on the activity of matrix metalloproteinase-2 (MMP-2) and MMP-9 and on the expression of glycosaminoglycans (GAG) in the rabbit cornea and crystalline lens., Methods: New Zealand rabbits were subjected to IR radiation for 4 months (chronic exposure to IR) or to normal light (control group). In experiments regarding acute exposure, animals were subjected to IR radiation or normal light for 12 h, in the presence of 0.1% diclofenac sodium (eye drops instilled in the right eye of animals) or saline (instilled in the left eye of animals). The cornea and lens were dissected away and homogenized. The activity of MMP-2 and MMP-9 was assayed by gelatine zymography. Total GAG were isolated from tissue specimens after lipid extraction and extensive digestion with pronase and DNase and characterized by treatment with GAG-degrading enzymes, followed by electrophoresis on cellulose acetate membranes., Results: Acute or chronic exposure to IR radiation induced the activity of MMP-2 in cornea and lens, whereas only acute IR radiation increased the content of heparan sulphate in crystalline lens. Local administration of diclofenac sodium did not prevent the above effects of acute IR radiation., Conclusions: The detrimental effects of excessive or prolonged exposure of the eyes to IR radiation are associated with induced activity of MMP-2 in cornea and lens and alterations in the content of heparan sulphate in lens. Thus, MMP and GAG may offer alternative targets for pharmacological intervention to confront ocular damages associated with IR radiation.

Purpose: The present study aims at determining whether enzymes of urea synthesis are expressed in the human lacrimal gland and in tissues of ocular surface (conjunctiva, cornea), to give evidence for the hypothesis that urea can be locally formed from ocular tissues and is important for the composition of the tear fluid., Methods: The presences of enzymes (arginase 1, 2 and agmatinase) that directly contribute to the formation of urea were investigated in the lacrimal gland and tissues of ocular surface by RT-PCR and immunohistochemistry. We collected tear fluid, aqueous humour, and blood samples from a total of 38 subjects, and tear fluid samples from a total of 78 subjects, with and without dry-eye syndrome (DES, keratoconjunctivitis sicca), and determined the urea concentration., Results: The enzymes arginase 1, 2 and agmatinase were expressed in all tissues examined except for arginase 1, which was not expressed in the cornea. There was no correlation of urea concentration in tear fluid with aqueous humour and blood plasma (r = 0.13, p = 0.58 and r = 0.45, p = 0.05 respectively). However, correlation of urea concentration between aqueous humour and blood plasma was highly significant (r = 0.7, p = 0.0001). The concentration of urea in the tear fluid of patients with DES compared to healthy control group was significantly reduced (p < 0.0001)., Conclusion: Enzymes that are directly involved in the formation of urea are expressed in ocular tissues. This may imply that in the ocular surface is a well-coordinated system of enzymes that can produce urea which might be independent of external urea supply.

The aim of this study is to determine the efficacy of a potent and specific vascular adhesive protein-1/semicarbazide-sensitive amine oxidase (VAP-1/SSAO) inhibitor, LJP 1207, as a potential antiangiogenic and anti-inflammatory agent in the therapy of corneal neovascularization. Corneal neovascularization was induced with intrastromal suturing in rabbits (n = 20). Topical treatment with VAP-1/SSAO inhibitor LJP 1207 (n = 5, 4 times a day), bevacizumab (n = 5, daily), their combination (n = 5) and vehicle only (n = 5, 4 times a day) were applied postoperatively for 2 weeks. The development and extent of corneal neovascularization were evaluated by digital image analysis. At the end of the observation period, the level of corneal and serum VAP-1/SSAO activity was measured fluorometrically and radiochemically. The corneal VAP-1/SSAO activity was significantly elevated in the suture-challenged vehicle-treated group (3,075 ± 1,009 pmol/mg/h) as compared to unoperated controls (464.2 ± 135 pmol/mg/h, p < 0.001). Treatment with LJP 1207 resulted in slower early phase neovascularization compared to vehicle-treated animals (not significant). At days 7-14, there was no significant difference in the extent of corneal neovascularization between inhibitor- and vehicle-treated corneas, even though inhibitor treatment caused a normalization of corneal VAP-1/SSAO activity (885 ± 452 pmol/mg/h). Our results demonstrate that the significant elevation of VAP-1/SSAO activity due to corneal injury can be prevented with VAP-1/SSAO inhibitor LJP 1207 treatment. However, normalization of VAP-1/SSAO activity in this model does not prevent the development of corneal neovascularization.

Purpose: In recent years many three-dimensional cornea models have been developed. However, they show poor collagen stability in the stroma. Transglutaminases (Tgases) are calcium-dependent proteins which play an important role in cross-linking of the corneal stroma. The purpose of this study was to find out whether it is possible to induce in vitro cross-linking of the stroma in an artificial hemicornea model with the help of Tgases., Materials and Methods: For the construction of the hemicornea, human SV40 adenovector corneal epithelial cells (HCE) and human SV40 adenovector corneal keratocytes (HCK) were cultivated. Confluent HCK cells were treated for 24 h with transforming growth factor beta (TGFb) 1, 2 and 3 at different concentrations as well as with other growth factors and the treated cells were compared to untreated cultivated cells. The quantification of the expression of the Tgases by HCKs was examined with the use of real time PCR, Western blot imaging and immunochemistry., Results: All concentrations of TGFbs used resulted in a significant increase of Tgase-mRNA, Tgase protein level and Tgase activity. The Tgases remained unaffected after treatment with other growth factors in comparison to untreated control cells. Treatment of the hemicornea with TGFb2 showed a very strong contraction and haze in comparison to the untreated hemicornea., Conclusion: It has been shown for the first time that TGFb induces a strong expression of Tgases in HCK cells. This effect caused an undesired contraction and haze of the human hemicornea model. Further research is necessary in order to find out whether the induction of Tgases in the HCK cells can be regulated without losing stability of the constructed hemicornea.

Background: At present there are no data in the literature on the expression of matrix metalloprotein-19 in the human cornea. The aim of this study was to analyze the expression of matrix metalloproteinase-19 in the human cornea and to investigate its potential role in corneal wound healing using a MMP-19 knock-out mouse model., Methods: A method with Western blotting and immunohistological staining for MMP-19 was performed using paraffin embedded human corneas. Excimer laser keratectomy was performed in wild type (wt) and MMP-19 knock-out (ko) mice and the rate of re-epithelialization was analyzed after 8 h and 18 h., Results: MMP-19 was strongly expressed in the human corneal epithelium mainly in the basal cell layer. MMP-19 was not expressed in the corneal stroma. In the mouse model the size of the corneal lesion after 8 h was 83% (wt) and 89.9% (ko) of the initial area (p=0.09). After 18 h the lesion was 17% (wt) and 13.3% (ko) of the initial area (p=0.01). Laminin-5 was expressed in the migrating epithelial cells with no differences between wild type and knock-out mouse., Conclusion: MMP-19 showed a strong expression in the basal cells of the human corneal epithelium. Corneal re-epithelialization was slightly faster in the MMP-19 knock-out mouse. No differences in the expression of laminin-5 could be detected.

Purpose: It has been shown that the treatment of keratoconus with riboflavin/ultraviolet A (UVA) causes significant stiffening of the cornea due to cross-linking. The aim of this study was to evaluate how deep the mechanical stabilization after collagen cross-linking could be shown biochemically., Method: Ten out of 20 enucleated porcine eyes were treated with riboflavin as a photosensitizer and UVA (370 nm, 3 mW/cm2, 30 min). The other 10 eyes served as controls. With a Microkeratom device, two flaps with a thickness of 200 microm and a diameter of 8 mm were cut off from each eye and put in a collagenase solution (NaCl plus collagenase A, 1:1). The surfaces of the flaps were measured digitally every day to characterize the dissolving behavior., Results: The resistance (regarding corneal collagen against enzymatic digestion) of the treated superficial flaps was considerably higher (p=0.001) compared to those that were cut secondarily and to the control flaps. But even the flaps from deeper layers showed a significant increase in resistance (p=0.02) compared with the untreated flaps. The half-life of the surfaces of the treated superficial flaps was 220 h; of those cut secondarily, it was 80 h. Both untreated flaps had a half-life of 50 h., Conclusions: The biochemical study showed that the treatment of the cornea with riboflavin/UVA leads to significant collagen cross-linking not only in the anterior slice of 200 microm but also in the following 200 microm. This locally limited cross-linking effect may be explained by the absorption behavior for UVA of the riboflavin-treated cornea; 65% of UVA irradiation is absorbed in the first 200 microm and only 25-30% in the next 200 microm. Therefore, deeper-lying structures and especially the endothelium are not affected.

Organotypic cornea equivalents are used as in vitro models for permeation studies. Many ophthalmic drugs are applied as ester prodrugs to achieve a higher bioavailability. The esterase activity of three corneal human cell lines (epithelial, stromal, endothelial cells) as well as of excised porcine cornea, human donor cornea and human cornea construct (HCC) was investigated and compared. Esterase activity was determined using p-nitrophenyl acetate and hydrocortisone acetate (HCA) as esterase substrates. Hydrocortisone acetate permeation across porcine cornea, human donor cornea and HCC was studied in vitro using Franz-diffusion cells. Corneal epithelial cells showed the highest esterase activity and only small differences to keratocytes and endothelial cells were detectable. The permeation barrier properties of the different corneal tissues were very similar in the case of HCA permeation whereas HCA metabolism rates were in the ranking order of porcine cornea > HCC > human donor cornea. Permeation and metabolism studies indicate that the in vitro permeation model HCC is able to adequately convert hydrocortisone acetate to hydrocortisone.

Background: The formation of free oxygen radicals has been demonstrated in the corneal tissue after 193 nm laser irradiation. Cornea has several defense mechanisms that protect against oxidative damage. One of them, glutathione peroxidase (GPx), catalyzes the destruction of hydrogen peroxide and lipid hydroperoxide. Selenium is a trace element which is incorporated into the selenoenzyme GPx. In the present study, the effect of excimer laser keratectomy on corneal GPx activities and aqueous humor selenium concentrations in rabbits was evaluated., Methods: Animals were divided into five groups, and all groups were compared: controls (group 1), after epithelial scraping (group 2), transepithelial photorefractive keratectomy(PRK; group 3), superficial traditional PRK (50 microm; group 4) and deep traditional PRK (100 microm; group 5). Corneal GPx activities were measured by a modification of the coupled assay procedure. Aqueous humor selenium concentrations were determined using hydride generation atomic absorption spectrometry., Results: Corneal GPx activities were significantly lower only in group 5 ( P<0.05), and the selenium concentration in the aqueous humor did not change in any group., Conclusion: Deep corneal photoablation inhibits GPx enzyme activities in the cornea. Therefore, antioxidants may be useful in reducing free radical-mediated complications after excimer laser corneal photoablation.

Background: Diabetic keratopathy, frequently observed after vitreous surgery, has been thought to be related to the aldose reductase-catalyzed reaction. However, few reports have been published on the chronological changes in polyol accumulation and the effect of the aldose reductase inhibitor in the corneal epithelium or endothelium of galactosemic rats. Consequently, the polyol accumulation in corneal epithelium and endothelium with stroma of 50% galactose-fed rats and the preventive effect of an aldose reductase inhibitor, SNK-860, were biochemically analyzed., Methods: Four-week-old male Sprague-Dawley rats were fed a 50% galactose diet without supplement or supplemented with a low (3 mg/kg b.w.) or high (30 mg/kg b.w.) dose of SNK-860 (Sanwa Kagaku Kenkyusho, Japan), or a normal diet. Polyol contents in the corneal epithelium or endothelium with stroma were individually measured using gas-liquid chromatography., Results: Polyol in corneal epithelia accumulated quickly in the 1st week, reached a maximum at the 3rd week of feeding and then gradually decreased. Low- and high-dose SNK-860 treatment significantly inhibited polyol accumulation in the epithelium and endothelium with stroma, respectively. Changing to the normal or SNK-860 supplemented diets significantly inhibited polyol accumulation., Conclusion: This finding indicates that oral administration of a new aldose reductase inhibitor, SNK-860, or systematic treatment of diabetes may be effective in preventing polyol pathway-induced corneal damage by quickly reducing the polyol level.

Background: A rat model of orthotopic corneal graft rejection was used to investigate the alterations in hydrolase activity within the corneal graft or within cellular infiltrates during acute rejection., Methods: The distribution of the lysosomal enzymes [acid phosphatase (AP), N-acetyl-beta-D-glucosaminidase (NAG), beta-glucuronidase (beta-Gluc), beta-galactosidase (beta-Gal), dipeptidylpeptidase II (DPPII)] and of the membrane-bound proteases [aminopeptidase M (APM), aminopeptidase A (APA), gamma-glutamyltransferase (GGT), alkaline phosphatase (ALP), dipeptidylpeptidase IV (DP-PIV)] were investigated by histochemical methods in the grafts at 3, 5, 8, 10 and 12 days following allogeneic transplantation. Serial sections of the grafts were also examined for RT1b, CD4, CD4+, CD8, CD11b/c and CD45, in order to determine hydrolase activity within infiltrating cells., Results: Allogeneic grafts were invaded by macrophages, CD4- and CD8-positive lymphocytes. In contrast, syngeneic grafts, performed as a control, contained occasional lymphocytes and focal aggregations of macrophages around suture sites. The allogeneic cellular infiltrate stained intensely for AP and ALP; moderately for beta-Gluc, NAG and beta-Gal; and mildly for GGT, DPPII and APM in grafts at all postoperative times. Serial sectioning indicated that the majority of the lysosomal hydrolases were located in macrophages; AP, APM and GGT were, however, observed in lymphocytes. Vessel ingrowth could be observed with enzyme staining for AP, beta-Gluc, NAG, ALP, APA and APM. Hydrolase activity in the corneal endothelium served as an indicator of endothelial function during the rejection process., Conclusion: Changes in normal hydrolase activities in corneal grafts in the rat model indicate decreasing corneal function during the rejection process. Hydrolases released from infiltrating cells contribute to the morphological disruption and, possibly, to graft rejection.

In order to evaluate the impact of tissue oxygenation on the distribution pattern of lactate dehydrogenase isoenzymes, activities of the isoenzymes were measured in microdissected samples of bovine tissue. A highly sensitive ultrathin-layer electrophoretic technique was used to determine the distribution pattern of lactate dehydrogenase isoenzymes in basal, intermediate and superficial layers of the epithelium of central and peripheral cornea and in the epithelium of the bulbar conjunctiva. Measurements revealed almost homogeneous intraepithelial distribution patterns of lactate dehydrogenase isoenzymes in both tissues. In the cornea the lactate dehydrogenase isoenzymes 4 and 5, which are regarded to be specialized for anaerobic glucose metabolism, were found to predominate. In the well-oxygenated conjunctival epithelium most of the activity could be ascribed to the lactate dehydrogenase isoenzyme 3. In contrast to the isoenzymatic activities, total activity of lactate dehydrogenase was inhomogeneously distributed; maximum activities were found in the basal layer of corneal epithelium and in the intermediate layer of conjunctival epithelium. The results indicate that oxygen supply is relevant rather for the intraepithelial distribution of total enzyme activity than for the expression of lactate dehydrogenase isoenzymes.

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 micrograms/ml) in the tear fluid. Middle activity of plasmin (1.0-2.0 micrograms/ml) was detectable when PMNs were present in the corneal stroma. High plasmin activity (2.0-3.0 micrograms/ml) correlated with corneal ulceration and vascularization.(ABSTRACT TRUNCATED AT 250 WORDS)

Rabbit eyes burned with 1 n NaOH were studied for enzymatic activity by biochemical methods. The activity of beta-N-acetylglucosaminidase (E.C. 3.2.1.50), one of the hydrolases, was found to be significantly increased in corneal epithelium and stroma 1, 2, 3, and 4 weeks after alkali burns. The increase in enzyme activity seemed to be in correlation with the polymorphonuclear leukocytes (PMNs) infiltration of the stroma, as found by histopathologic studies. Therefore the PMNs might be the source of the increasing activity of this enzyme after alkali burns.

Lactate dehydrogenase (LDH) isoenzymes of rabbit lens and other intraocular tissues are separated by thin-layer isoelectric focusing and localized as discrete groups of multiple bands with defined isoelectric points after staining by the tetrazolium method. In the rabbit lens parts, the predominant isoenzymes are LDH-4 and LDH-5. The bands show microheterogeneity, are composed of 2-4 subcomponents, and the pattern shows a distribution of the liver type. The activity of the LDH-4 decreases and that of LDH-5 increases in an order given by the equator, anterior and posterior cortex, and nucleus. LDH-3 remains almost constant in all lens parts. LDH-4 is composed of two subcomponents, one of which, the most cathodic with higher isoelectric point, is almost absent in the lens nucleus. Of the LDH localized in the rabbit intraocular tissues, only the retina shows a pattern of five isoenzymes also of the liver type. In all intraocular tissues LDH-3, -4, and -5 are very prominent, show also microheterogeneity of their isoenzyme bands, and are each composed of 4-6 subcomponents. LDH-1 and -2 show only one isofocused component. Species specificity is shown of the LDH isoenzymes in the rabbit, mouse, dog, and calf lens.

Alkaline and acid phosphatases were studied in the rabbit cornea following acetone and ethanol exposure to the eye. Changes in enzyme activities were investigated in homogenates of epithelium and stroma quantitatively and in frozen cryostat sections on days 1, 4, 7, 14, and 28. Biochemical and histochemical findings showed a remarkable increase in alkaline phosphatase of the epithelium beginning on day 7. This activation persisted until day 28 after instillation of both noxae with maximum activity on day 14. However, acetone was proved to be significantly more effective than ethanol. In addition, a different topochemistry of alkaline phosphatase was found in the epithelium of treated corneas, i.e., enzyme activity was observed not only superficially but in all epithelial layers of the cornea as compared to a normal one. The effect of acetone and ethanol on a regenerating corneal epithelium is discussed.

Isolated rabbit corneae were stored at a temperature of 4 degrees C in McCoy's 5a medium (modified) containing 5% dextran T 40 and gentamycin sulphate (250 mug/ml) for a varied number of days. The endothelium of the corneae was examined by light- and electron-microscopic histochemical methods for ATPase, TPPase, and SDH. The activity of each enzyme in the endothelia, stored for 6 days, was found to resemble the enzyme activities in the fresh corneal endothelium. By 8 days, the SDH activity was lowered and specific staining for ATPase and TPPase was decreased. In the 12 day-stored endothelium, the SDH and TPPase activities could not be detected. From these results, it was gathered that the isolated cornea stored in the medium for 6 days may be used in keratoplasty.

In rabbits inflammation of the cornea was induced by intrastromal injection of horse serum. Between 2 and 4 weeks after injection, infiltration of the cornea with leukocytes and neovascularization could be observed. During this period, the rabbits were killed and their corneas analyzed for protein, alkaline phosphatase, acid phosphatase, N-acetyl-beta-D-glucosaminidase, and lactate dehydrogenase. The enzyme activities in the inflamed corneal stroma reflect the high lysosomal activity, which probably originates from the leukocytes. The enzyme activities in the epithelium indicate that the tissue is abnormal and undergoing repair processes.

Hyaluronidase activity in human corneoscleral tissues was biochemically studied. The corneoscleral junction, particularly the inner layer, showed higher activity than other corneoscleral tissues. The possible role of lysosomal hyaluronidase in the trabecular meshwork is discussed.

The distribution of alkaline phosphatase was investigated in corneas of various animals by means of histochemical and biochemical methods. Special attention was paid to keratocytes that proved to be positive when a proper substrate and technique were used even if the activity differed according to animal species. Naphthol-AS-MX-phosphatate with Variamine-Blue-RT salt in the simultaneous azocoupling method was the most sensitive substrate; less suitable were naphthol-AS-phosphate, and particularly l-naphthyl-phosphate with Fast-Blue-BB-salt in the same method. Keratocytes were completely negative with beta-glycerophosphate in the Gomori technique. Contrary to keratocytes, epithelial and endothelial cells were without substrate predilection. The results of both approaches showed a wide inter-species variability. The overall activity of alkaline phosphatase detected histochemically and its activity in the unsedimentable fraction of whole corneas determined biochemically was in good agreement. The highest activity was found in the calf cornea; enzyme levels decreased gradually in the bovine, rabbit, dog, sheep, and pig cornea.

The localization of CA activity in the rabbit cornea was investigated using a modified Hansson's method. The reaction products indicating CA activity was observed in the lateral membrane of endothelial cell, but not in the apical membrane. In cytoplasm, Golgi complex, and mitochondria of the endothelial cells no specific reaction product could be found. In epithelium, stroma, and Descemet's membrane, no reaction product was seen. The specific staining was inhibited in the control group which was treated with 10(-4)M sodium acetazolamide.

The thiocholine method for the histochemical detection of cholinesterases according to Karnovsky-Roots was adapted for unfixed cryostat sections by addition of the agar solution to the incubation mixture and by using the semipermeable membrane interposed between the section and the incubation medium. The procedure prevents the leakage of the enzyme activity of the section and is suitable for tissues where the cholinesterase activity is low.

The activities of acid phosphatase, beta-glucuronidase, beta-galactosidase, and N-acetyl-beta-D-glucosaminidase were investigated in the normal rabbit cornea. For all spectrophotometric assays, appropriate p-nitrophenyl derivates were used. Only beta-glucuronidase were determined employing phenolphthalein glucuronid as a substrante. Acid phosphatase revealed the highest activity, followed by N-acetyl-beta-D-glucosaminidase and beta-galactosidase. In the case of beta-glucuronidase the lowest activity was found. The results on the rabbit cornea are compared with those on some other tissues described in the literature. Correlation of biochemical data and histochemical findings in the same species is briefly discussed.

An experimental apparatus, which uses freshly collected, nondenatured rat tail tendons as substrate against crude corneal collagenase from alkali-burned rabbit corneas and clostridiopeptidase A, is introduced. The apparatus makes it possible to compare the collagenolytic activity of the two enzymes directly on intact connective tissue similar to intact corneal tissue. It was found that the two enzymes were able to reduce the tensile strength of rat tail tendons to less than 100 g in less than 5 h. The two enzymes attacked the tendons in structurally the same manner, estimated from a statistical model. The conclusion drawn is that crude corneal collagenase can degradate intact connective tissue indicating that it can attack the intact cornea.

Alkali-burnt corneas of the rabbits with 1 N NaOH were studied periodically for enzymatic activities by biochemical methods. There was significant increase of aldolase (ALD) activity both in corneal epithelium and stroma 1, 2, 3 and 4 weeks after alkali burns. Glucose-6-phosphate dehydrogenase (G6PD) activity was significantly decreased in epithelium and was absent in stroma. Thus the breakdown of glucose would be present preferably in the Embden-Meyerhoff pathway instead of the pentose phosphate shunt. Lactate dehydrogenase (LDH) activity of corneal epithelium and stroma was significantly decreased 1, 2, 3, and 4 weeks after alkali burns and the possible pathway of glycolysis might channel to citric acid cycle, in which malate dehydrogenase (MDH) could indicate the important role in this pathway.

The sodium-potassium activated adenosine triphosphatase (NaKATPase) activity of the rat cornea was investigated histochemically using a Pb2+-precipitation technique in which adenosine triphosphate (ATP) is used as substrate and two methods for potassium-dependent para-nitrophenyl-phosphatase (K-NPPase) activity. With all the three techniques used it was demonstrated that the sodium-potassium-activated adenosine triphosphatase (NaK-ATPase) activity is localized in the cell membranes of the endothelium whereas a much weaker activity was observed in the epithelium. When the Pb2+-technique was used, the epithelial cell membranes showed a weaker reaction in the presence of ouabain. This activity was only Mg2+-dependent and was presumably due to an Mg2+-dependent ATPase. The validity of the histochemical techniques for NaK-ATPase activity is discussed. The results emphasize the importance of the endothelium as the main site of Na+ transport in the cornea. Small amounts of the enzyme are also present in the epithelium, which seems to be rich in Mg2+-ATPase. Provided that careful controls are performed, all the methods give consistent results in the cornea.

Activities of alkaline and acid phosphates were investigated in rabbit corneas after a complete epithelial denudation in vivo (limbus to limbus). Dynamics of enzymatic changes during corneal healing were followed quantitatively in homogenates of regenerated epithelium and stroma (denuded cornea) and in cryostate frozen sections on days 1, 4, 7, 14, and 28. Biochemical and histochemical findings at these times showed a different response in each enzyme. Acid phosphatase displayed a gradual increase of activity in epithelium as well as in stroma; on day 28 after injury its content was normal. In contrast, alkaline phosphatase showed delayed activity during the repair process, and even a month after de-epithelization was still subnormal, particularly in superficial layers of epithelium.

In order to assess the extent of injury and the response of the cornea to alkali burns, NaOH in concentrations of 0.5, 0.25, 0.1, 0.05, and 0.01 N was applied to rabbit eyes and the histologic and metabolic changes studied. The activities of alkaline and acid phosphatases in homogenates and in cold microtome sections were examined on days 1, 4, and 7 after injury. At all time intervals 0.5 N and 0.25 N NaOH induced a remarkable decrease in enzyme activities. On the other hand, after 0.05 N and 0.01 N NaOH only very slight changes were observed. Using 0.1 N NaOH, both phosphatases decreased on day 4 after treatment and acid phosphatase reached normal values in a week, whereas alkaline phosphatase increased with a maximum on day 7. Its role in synthetic processes during corneal regeneration is discussed. Both histologic and metabolic patterns in the experimentally burned cornea were shown to be a function of NaOH concentration and the duration of contact. The process of re-epithelization of the cornea during healing after 0.1 N NaOH for 1 min is described.

In vitro study of the uptake of horseradish peroxidase (HRP) by the corneal endothelium was carried out on rabbit corneae which were stored in a preserving medium for various periods. The stored corneae were incubated in HRP solution with or without adenosine triphosphate (ATP). The number of pinocytotic vesicles decreased as the storage time was prolonged. Addition of ATP to the HRP solution led to an increase in the pinocytotic vesicles in all storage periods. The pinocytosis in the 12-day stored corneal endothelium was only observed when the tissue was incubated in an HRP solution containing ATP. From these results, it was concluded that the viability of the stored corneae can be evaluated with uptake of HRP by the endothelial cell, and that the addition of ATP leads to an enhancement of pinocytotic activity in the endothelial cell.

In the rabbit and bovine cornea the activity of alkaline phosphatase using histochemical as well as biochemical methods was investigated. Biochemically the enzyme activity was studied in separated corneal layers. In the histochemical investigation the best results were obtained in cryostat sections using the azocoupling method with naphthol AS-MX phosphate and Variamine Blue RT Salt. The enzyme activity was found not only in the epithelium and endothelium (as was described previously) but even in keratocytes. The mutual relation of activities in the epithelium and in keratocytes differed in both species. The overall activity found by histochemical methods is in good agreement with the biochemical determination of alkaline phosphatase (p-nitrophenyl phosphate as the substrate). Besides the histochemical approach shows an uneven distribution of alkaline phosphatase activity in individual cells which cannot be assessed by the biochemical determination.

The distribution of carbonic anhydrase (CA) in the eye of man and three species of monkeys was studied by light and electron microscopy using the histochemical method of Hansson. Carbonic anhydrase staining was found in the corneal endothelium. In monkeys the endothelial cells covering the inner surface of the operculum were also stained, whereas in the human the staining stopped at Schwalbe's line. In the trabecular meshwork no cells exhibited CA staining. The iris dilator muscle showed some staining, while no clear staining was found in the pigment epithelium (PE). In the ciliary body both the PE and the non-pigmented epithelium (NPE) displayed CA staining, most prominently in the basal and lateral membranes but also discernible in the cytoplasm. The staining of the NPE (but not the PE) showed clear-cut regional differences, and the presence of CA coincided with morphological indicators of secretory activity. Heavy staining was found in the capillaries under the ciliary epithelium. In the pars plana many capillaries showed staining of only that part of the circumference of the vessel wall which faced the epithelium. Unstained segments were also found in many choroidal capillaries under the pigmented epithelium of the retina. In the retina itself CA stain was found in the pigmented epithelium, in the Müller cells and in capillaries supplying the inner retina (only monkey retina was studied).(ABSTRACT TRUNCATED AT 250 WORDS)

A quantitative study was carried out on the lysosomal enzyme activities of the bovine corneal endothelium-Descemet's membrane preparation. The corneal endothelium and Descemet's membrane were peeled off together. Cathepsin D was assayed using hemoglobin as substrate; N-acetyl-beta-D-glucosaminidase, beta-glucuronidase, acid phosphatase, and alpha-mannosidase were also examined using p-nitrophenyl derivatives as substrate. The proportions of N-acetyl-beta-D-glucosaminidase, cathepsin D, and beta-glucuronidase of the Descemet's membrane-endothelium complex were particularly high: 11.5%, 12.6%, and 12.5% of the whole cornea, respectively. Corneal endothelial cells also showed high activities of acid phosphatase and alpha-mannosidase (3.8%, and 5.0% of the whole cornea, respectively), while the protein and DNA contents were 0.5% and 0.5% in the complex. Lysosomal enzyme activities in the complex were also compared with those in other ocular tissues and were determined by the same methods at the same time.

Activities of acid phosphatase, beta-glucuronidase, N-acethyl-beta-D-glucosaminidase and acid beta-galactosidase were investigated histochemically in rabbit corneas. Frozen sections after block fixation in cold 4% formaldehyde with 1% CaCl2 followed by washing in cold physiological saline as well as cold microtome sections of corneas quenched in petroleter chilled with acetone-dry ice mixture, transferred to nonprecooled slides or semipermeable membranes were used. Standard aqueous media were employed in the case of free-floating frozen sections of fixed corneas as well as of cold mictrotome sections (postfixed in cold 4% formaldehyde). Agar media were used in connection with the technic of semipermeable membranes. Gomori method (in the case of acid phosphatase), simultaneous azocoupling methods (substrates derivated of naphthol-AS-BI with hexazonium-p-rosanilin) in the case of acid phosphatase, beta-glucuronidase and N-acetyl-beta-D-glucosaminidase and the indigogenic method in the case of acid beta-galactosidase were applied. Enzyme activities in sections of fixed corneas were minimal in comparison with those in cold microtome sections of unfixed material revealed particularly with the technic of semipermeable membranes which is to be preferred. This technic is recommended in studies concerned with lysosomal enzymes in the cornea, particularly in keratocytes. All enzymes investigated were present in corneal epithelium, keratocytes and endothelium. Acid phosphatase displayed the highest activity followed by beta-glucuronidase and acetyl-beta-D-glucosaminidase. The activity of beta-galactosidase was the lowest. For the demonstration of activities in keratocytes sections parallel to the surface are very suitable. In these sections enzyme activities were demonstrated in small granules (apparently lysosomes) present in the central part of their cytoplasm as well as in projections. Diffuse staining was also seen, being the highest in the case of acid phosphatase.

The activities of acid phosphatase, N-acetyl-beta-D-glucosaminidase, alpha-mannosidase, alpha-fucosidase, beta-glucuronidase, arylsulfatase, and cathepsin D were biochemically investigated in the bovine cornea by separating the tissue into two layers, epithelium and stroma-endothelium. Acid phosphatase, alpha-mannosidase, alpha-fucosidase, and arylsulfatase disclosed much higher activities in the epithelial layer than in the stroma-endothelial layer. The other enzymes showed little difference in enzyme activity between the two layers.

Cholinesterase (ChE) activities of the rat cornea were demonstrated histochemically by using both light and electron microscopes. Acetylcholinesterase (AChE) reaction was localized in the axolemma of the nerves in the corneal stroma. The epithelial cell membranes and the intraepithelial nerve endings also showed AChE reaction. Non-specific cholinesterase (NsChE) activity was observed only in the endothelial cell membranes. Cervical sympathectomy, ciliary ganglionectomy and stereotactic coagulation of the ophthalmic division of the trigeminal nerve were performed in order to study the routes of the AChE-containing nerves to the cornea. The disappearance of AChE-containing nerves was observed only after ophthalmic neurotomy. It is suggested that the AChE-containing nerves are distributed to the rat cornea exclusively via the ophthalamic nerve. They seem to be sensory nerves.

The distribution of activities of membrane aminopeptidases (aminopeptidase M (APM), aminopeptidase A (APA), dipeptidyl peptidase IV (DPP IV), gamma-glutamyltransferase (GGT) and lysosomal exopeptidases (dipeptidyl peptidase I (DPP I), dipeptidyl peptidase II (DPP II)) was investigated in rabbit, ox and pig corneas. Cryostat sections of snap-frozen corneas treated with chloroform-acetone (4 degrees C) were used for the demonstration of membrane-bound enzymes and sections of corneas fixed in 4% paraformaldehyde (4 degrees C) for the demonstration of lysosomal enzymes. In activities of proteases species differences were found. The rabbit cornea was most active, followed by ox and pig corneas. Individual corneal layers reacted differently. Of membrane proteases a high APM activity was found in keratocytes, whereas epithelium and endothelium were negative. On the other hand, APA and GGT were active in the epithelium and endothelium. Their activities in keratocytes were less pronounced. DPP IV activity was demonstrated in some keratocytes beneath the epithelium only. Lysosomal enzymes DPP I and DPP II were active in all corneal layers. The epithelium displayed the highest activity. Differences in activities in the centro-peripheral and epithelio-endothelial directions were found. DPP I, DPP II, and APM were most active in the limbal region in all corneal layers.

Localization of NDPase and TPPase activities in the rabbit corneal endothelium was investigated by cytochemical methods. After incubation at both pH 7.0 and 8.0, the NDPase activity was detected in cisternae of ER, nuclear envelope, and Golgi saccules, while the TPPase activity was demonstrated only in Golgi saccules. The reaction products of both enzymes were decreased when incubated at pH 8.0. The NDPase activity was inhibited by uranyl nitrate but not by sodium fluoride, while the TPPase was inhibited by fluoride but not by uranium.

In alkali burned rabbit corneas activities of beta-glucuronidase, N-acetyl-beta-D-glucosaminidase and acid beta-galactosidase were studied histochemically in various time intervals after the traumatization. The technic with semipermeable membranes was employed. Within four days after the injury enzyme activities in the traumatized area were almost lacking. The corresponding activities in the unaffected part of the cornea were within the norm. On the 7th day enzyme activities were on an increase (but still subnormal) in the traumatized area. This area was surrounded by a zone of keratocytes with high levels of enzyme activities. This was particularly remarkable in keratocytes subjacent to the epithelium. The activation of all enzymes studied was present in the basal layer of the epithelium and in the endothelium as well. On the 14th day enzyme activities in the traumatized area were nearly restored and on the 32nd day they could not be distinguished from the normal cornea. Beta-galactosidase displayed a relatively maximal increase in the activity of all enzymes investigated.

The authors studied the release of fibrinolytic activators from in vitro cultures of human and animal cornea with the following main findings: (1) the epithelium is the main activator source; (2) the addition of tripeptide, salicylate, chloroquine and phorbolester to the culture medium increases the fibrinolytic activity of the cultures; (3) corticosteroids depress the fibrinolytic activity; (4) explants of human conjunctiva also release fibrinolytic activators. The pathophysiological role of this activator is unknown. Hypothetically, besides preventing fibrin deposition on the surface of the eye, this activator may also stimulate leucocyte migration, activate procollagenase, protect the endothelial pump, and regulate vascular permeability and angiogenesis in the cornea.