Histochemical study of alkali-burned rabbit anterior eye segment in which severe lesions were prevented by aprotinin treatment

Activities of different enzymes (acid glycosidases, phosphatases, Na+ - K+ -dependent ATPase, proteases, dehydrogenases) and acid glycosaminoglycans were studied by histochemical methods in sections of rabbit anterior eye segments after experimental alkali burn and treatment with aprotinin, an inhibitor of plasmin and other serine proteinases. Solutions of sodium hydroxide (0.25-1.0 M) were applied on corneas using 12-mm-diameter plastic tube for 15-60 s. After wiping with cotton and rinsing with tap water aprotinin solutions were applied in saline (in experimental animals) and saline (in control animals) dropwise in 12-h intervals for a month. Within the first two weeks aprotinin was used at a concentration of 5000 IU/ml. During the subsequent two weeks the aprotinin concentration was reduced to 2500 IU/ml. Striking differences in enzyme activities and in the healing between treated and untreated eyes were found. Without aprotinin, ulcers developed in most corneas within 3 weeks and plasmin was regularly demonstrated in tears and in the aqueous. When aprotinin treatment was started within 24 h after the burn, the number of enzymatically active inflammatory cells was significantly lower, not only in the cornea itself but also in the whole anterior eye segment. With aprotinin treatment no ulcerations and no plasmin in tears and the aqueous were observed and the corneas healed within a month. The healing process started from the zone of enzymatically activated corneal cells in the unburned zone at the corneal periphery. In the regenerating epithelium and endothelium high activities of Na+ -K+ -dependent ATPase, gamma-glutamyltransferase, lactate and succinate dehydrogenases appeared very soon.(ABSTRACT TRUNCATED AT 250 WORDS)     

Histochemical changes in the rabbit cornea and plasmin activity in the tear fluid during contact lens wear. Favourable influence of protease inhibitors (aprotinin,PC5, elastatinal)

Summary 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 g/ml) in the tear fluid. Middle activity of plasmin (1.0–2.0 g/ml) was detectable when PMNs were present in the corneal stroma. High plasmin activity (2.0–3.0 g/ml) correlated with corneal ulceration and vascularization. The occurrence of both — plasmin activity and corneal disturbances was highly dependent on the water content of SCL (which goes parallel with oxygen permeability), duration of SCL wear, mechanical stress, and bacterial contamination. Mechanical irritation is considered to be the main factor leading to the appearance of plasmin activity in the tear fluid. The local application of aprotinin which inhibits plasmin and some other serine proteases, enables us to prolong the harmless wear of SCLH (approximately one week). The combination of aprotin-in with leukocyte elastase inhibitors (elastatinal and particularly PC5), prevents ulceration of the cornea and inhibits corneal vascularization after SCLL wear. Vascularization of the cornea does not occur if protease inhibitors are combined with flurbiprofen, an anti-inflammatory drug of cyclooxygenase pathway of arachidonic acid. Protease inhibitors also improved the course of bacterial keratitis.     

The histochemical pattern of mechanically or chemically injured rabbit cornea after aprotinin treatment: relationships with the plasmin concentration of the tear fluid

Summary Plasmin, a serine protease, was recently found to be involved in corneal ulcerative processes in humans and rabbits. In our experiments, plasmin activity was found in the tear fluid after mechanical and chemical damage of the rabbit cornea, such as de-epithelization and burning with alkali. The plasmin concentrations in the tear fluid were dependent on the severity of injury. The highest plasmin activity (2.0–3.0 g ml^–1) occurred after severe alkali damage to large areas of the cornea, and the lowest activity (0.4–1.0 g ml^–1) after mechanical injury (de-epithelization).Plasmin concentrations up to 1.0 ml^–1 were associated with increased activities of lysosomal hydrolases in epithelial cells and keratocytes beneath the epithelium. Plasmin activities increased as the inflammatory reaction developed. When plasmin activity in the tear fluid was higher than 1.0 g ml^–1, inflammatory cells were found in the corneal stroma. Levels of 1.5–2.0 g ml^–1 were connected with higher numbers of inflammatory cells (particularly polymorphonuclear leukocytes) with increased activities of lysosomal hydrolases. Very high plasmin activities (2.5–3.0 g ml^–1) accompanied corneal ulcerative processes.The local application of aprotinin (Trasylol, Bayer), an inhibitor of plasmin, and also of some other proteases, was found to be necessary for the healing of severe corneal injuries in which highly elevated plasmin activity in the tear fluid and inflammatory cellulization of the cornea occurred (severe damage). It was beneficial in cases in which medium plasmin activity occurred in the tear fluid and inflammatory changes in the cornea were not too extensive. If used very early after injury, aprotinin prevents the appearance of high plasmin activity in the tear fluid, reduces the invasion of inflammatory cells into the corneal stroma, and accelerates the healing. Even the corneal transparency is restored in many cases.     

Disturbances in the rabbit cornea after short-term and long-term wear of hydrogel contact lenses

Summary The influence of soft contact lenses (SCL) with low (37%, L) and high (65%, H) water content on rabbit corneas was investigated. The lenses were worn continuously for 1, 2, 4, 7, 10, 14, 21 or 28 days. The changes in corneal transparency, hydration and enzyme activities were studied. A slight change in corneal transparency due to higher hydration caused by a decreased activity of Na⁺–K⁺-dependent adenosine triphosphatase (Na⁺–K⁺-ATPase) in the corneal endothelium is followed by a decrease in the activity of -glutamyl transferase (GGT). Slight morphological disturbances appear within 4 days in animals wearing SCL (L). SCL (H) produce similar changes one week later. Subsequently, the corneal epithelium becomes thinner and changes in the size of corneal endothelial cells are obvious. Disturbances of enzyme activities in cells of all corneal layers are present. In the epithelium highly increased activities of acid glycosidases, acid phosphatase, and dipeptidyl peptidase I and II, in keratocytes decreased activities of alkaline phosphatase and GGT, and in the endothelium decreased activity of Na⁺–K⁺-ATPase and GGT were found. These changes are more severe after SCL (L). In this case, inflammatory cells displaying high activities of lysosomal hydrolases appear in the anterior part of the stroma during the 3rd and 4th weeks and local degradation of glycosaminoglycans and proteins takes place. In contrast, after SCL (H) a remarkable thinning of the corneas was observed during extended wear, accompanied by decreased stainability of stromal glycosaminoglycans and highly decreased enzyme activities in keratocytes. The histochemical methods proved very useful in the assessment of tesions caused by a continuous wear of SCL.     

Secretory capacity of the lachrymal salt gland of hatchling sea turtles,Chelonia mydas

Summary The lachrymal salt glands ofChelonia mydas were functional when hatchlings emerged from the nest. Osmotic concentrations up to 720 mosmol kg^–1 were recorded in spontaneously produced tears (salt gland secretions). When injected with a Na⁺ load (1500–2700 mol (100 g)^–1) newly emerged hatchlings produced tears ranging in osmotic concentration from 1000–1900 mosmol kg^–1 with Na⁺ secretion rates from single glands of 200–475 mol (100 g·h)^–1. In these circumstances the rate of sodium excretion, via the salt glands, was equivalent to the sodium content of 0.2 to 0.5 ml of sea water per hour. Since the apparent drinking rate of hatchlings within the first two days of entering sea water was approximately 0.5 to 1.7 ml per day, the excretion of Na⁺ imbibed by drinking is well within the secretory capacity of the lachrymal salt glands.In feeding hatchlings extraordinarily high Na⁺ secretion rates were induced by Na⁺ loading. Hatchlings which were loaded with Na⁺ by injection (1500–5400 mol (100 g)^–1) produced tears having osmotic concentrations between 1500 and >2000 mosmol kg^–1. The Na⁺ secretion rates from single glands were 750–4185 mol (100 g·h)^–1 with extremely high short term rates of 10700 mol (100 g·h)^–1 (50 mol min^–1 for 28 g hatchlings).In terms of gland mass the highest long term secretion rate translates into 21 mmol of Na⁺ per gram of salt gland per hour and is the highest secretion rate yet recorded for a reptilian salt gland. This rate is almost three times the highest rate recorded for sea snakes (8 mmol g·h^–1) and is similar to rates commonly observed in avian salt glands (25 mmol g·h^–1).Secretion by the lachrymal salt glands was initiated by increased blood concentrations of Na⁺ or K⁺, K⁺ being as effective as Na⁺ but with the composition of the teras being virtually unchanged compared to tears from Na⁺ stimulated hatchlings. Preliminary experiments indicated that secretion was not initiated by increased Cl^– concentration in the blood or by increased volume or osmotic concentration of the blood.Abbreviation O.P. osmotic pressure     

Transient outwardly rectifying potassium channel in the rabbit corneal endothelium

Summary Ionic currents from freshly dissociated rabbit corneal endothelial cells were examined using patch-clamp technology and a perforated patch technique. Whole-cell current recordings revealed a transient outward K⁺-selective current that was blockable in a dose-dependent manner by 4-aminopyridine (4-AP) and quinidine. This current is similar to the A-type current present in many excitable cells and is the first reported instance of such a current in any epithelial cell type. In addition to the transient current, an outwardly rectifying nonselective cation current was also observed. This current is also blocked by quinidine.To examine the possible role of these currents in the stromal volume regulatory function of the endothelium, corneas were perfused under a specular microscope with a glutathionebicarbonate Ringer's solution (GBR) or GBR plus either 1 mM quinidine or 10 mM 4-AP. For quinidine perfusions, control corneas swelled at a rate of 6 m/hr, while quinidine-perfused corneas swelled at a rate of 48 m/hr. For 4-AP perfusions, control corneas deswelled at a rate of –2 m/hr, while 4-AP perfused corneas swelled at a rate of 24 m/hr. One possible mechanism of the stromal swelling induced by these K⁺ channel blockers may be the result of loss of the K⁺ recycling pathway necessary for proper Na⁺/K⁺ ATPase function.We would like to thank Dr. William Bourne for the use of his specular microscopy corneal perfusion apparatus and Helen Hendrickson for her technical assistance. This work was supported by NIH grants EY06206, EY03282, EY06005, and an unrestricted award from Research to Prevent Blindness.     

Evaluation of the Shape Symmetry of Bilateral Normal Corneas in a Chinese Population

Purpose

To investigate the bilateral symmetry of the global corneal topography in normal corneas with a wide range of curvature, astigmatism and thickness values

Design

Cross-Sectional Study

Methods

Topography images were recorded for the anterior and posterior surfaces of 342 participants using a Pentacam. Elevation data were fitted to a general quadratic model that considered both translational and rotational displacements. Comparisons between fellow corneas of estimates of corneal shape parameters (elevation, radius in two main directions, R_x and R_y, and corresponding shape factors, Q_x and Q_y) and corneal position parameters (translational displacements: x₀, y₀ and z₀, and rotational displacements: α, β and γ) were statistically analyzed.

Results

The general quadratic model provided average RMS of fit errors with the topography data of 1.7±0.6 µm and 5.7±1.3 µm in anterior and posterior corneal surfaces. The comparisons showed highly significant bilateral correlations with the differences between fellow corneas in R_x, R_y, Q_x and Q_y of anterior and posterior surfaces remaining insignificantly different from zero. Bilateral differences in elevation measurements at randomly-selected points in both corneal central and peripheral areas indicated strong mirror symmetry between fellow corneas. The mean geometric center (x₀, y₀, z₀) of both right and left corneas was located on the temporal side and inferior-temporal side of the apex in anterior and posterior topography map, respectively. Rotational displacement angle α along X axis had similar distributions in bilateral corneas, while rotation angle β along Y axis showed both eyes tilting towards the nasal side. Further, rotation angle γ along Z axis, which is related to corneal astigmatism, showed clear mirror symmetry.

Conclusions

Analysis of corneal topography demonstrated strong and statistically-significant mirror symmetry between bilateral corneas. This characteristic could help in detection of pathological abnormalities, disease diagnosis, measurement validation and surgery planning.     

Application of Preserved Human Amniotic Membrane for Corneal Surface Reconstruction

Objective: To evaluate the efficacy of preserved human amniotic membrane transplantation for reconstruction of the corneal surface diseases. Methods: Preserved human amniotic membrane transplantations were performed in 84 eyes of 78 patients for corneal surface reconstruction. The indications were limbal stem cell deficiency from Steven–Johnson syndrome, chemical burn and herpes keratitis (27 eyes), bullous keratopathy (26 eyes), persistent epithelial defect and dellen (17 eyes), band keratopathy (11 eyes), preparing for prosthesis (1 eye), corneal ulcer (1 eye) and acute chemical burn (1 eye). Results: Success was noted in 83.3% (70/84) eyes, partial success in 13.1% (11/84) eyes, and failure in 3.6% (3/84) eyes for an average follow-up of 10.5 months (3 – 29 months). No patient developed major immediate post-operative complications. Conclusion: Amniotic membrane transplantation can reduce inflammation, promote corneal epithelial healing, and decrease irritation in corneal surface problems.     

The stimulation of chloride transport by prostaglandins and their interaction with epinephrine,theophylline, and cyclic AMP in the corneal epithelium

Summary Prostaglandins (E₁, E₂ and F₂) stimulated the chloride transport of the frog corneal epithelium with maximal effects at 10^–5 m in the aqueus side. This stimulation does not occur in Cl-free solutions and the net³⁶Cl flux increased proportionally to the short-circuit current. Polyphloretin phosphate (PPP) and diphloretin phosphate (DPP) inhibited the response if added within 3 min before PGE₁. The maximal response to epinephrine 10^–5 m and dibutyryl cyclic AMP 10^–3 m was not changed by further addition of prostaglandins, but these drugs produced their full effect when administered at the peak of the response of prostaglandins. The maximal response to theophylline 10^–5 m was increased by PGE₁. PPP and DPP did not modify the response to epinephrine. Prostaglandin stimulation of the chloride transport was accompanied by increased light transmission through partially opaque corneas. The known release of prostaglandins in the aqueous humor can be associated to a direct action on the corneal epithelium manifested in the activation described herein.     

Development of corneal transparency in embryonic chick: influence of exogenous thyroxine and thiouracil on structure, water and electrolyte content.

Thiouracil and thyroxine (T₄) were injected onto the chick chorioallantois at various developmental stages to study their effects on corneal cellularity, dehydration and structure. Corneas were excised 2–9 days after treatment for histological examination and for analyses of water content, sodium concentration [Na⁺] and potassium concentration [K⁺]. Untreated chick corneas showed that water content and [Na⁺] decreased with advancing embryonic age, while [K⁺] increased up to stage 42 and then rapidly declined. Corneas from embryos injected with 10 mg thiouracil at stage 36 had a significantly reduced [K⁺] at stages 40 and 42. Corneas at stages 40, 42 and 45 had a significantly elevated water content when compared with controls. Injection of 15 μg of T₄ prior to stage 36 or at or after stage 40 did not produce significant changes in corneal water and ionic content compared with controls; injection of 15 μg of T₄ during the cell proliferation period of corneal development (stages 36–40) produced a significant increase in [K⁺]. Similar results were obtained in corneas from embryos injected with 1 μg of T₄. Total corneal thickness was increased in thiouracil treated corneas, and decreased in T₄ treated corneas. Epithelial growth was markedly decreased with thiouracil treatment, while T₄ had little effect. It is likely that thiouracil treatment decreases cell division in the cornea, and prevents formation of the epithelial barrier, whereas T₄ accelerates these processes.     

Distinct Roles for Dectin-1 and TLR4 in the Pathogenesis of Aspergillus fumigatus Keratitis

Aspergillus species are a major worldwide cause of corneal ulcers, resulting in visual impairment and blindness in immunocompetent individuals. To enhance our understanding of the pathogenesis of Aspergillus keratitis, we developed a murine model in which red fluorescent protein (RFP)-expressing A. fumigatus (Af293.1RFP) conidia are injected into the corneal stroma, and disease progression and fungal survival are tracked over time. Using Mafia mice in which c-fms expressing macrophages and dendritic cells can be induced to undergo apoptosis, we demonstrated that the presence of resident corneal macrophages is essential for production of IL-1β and CXCL1/KC, and for recruitment of neutrophils and mononuclear cells into the corneal stroma. We found that β-glucan was highly expressed on germinating conidia and hyphae in the cornea stroma, and that both Dectin-1 and phospho-Syk were up-regulated in infected corneas. Additionally, we show that infected Dectin-1^−/− corneas have impaired IL-1β and CXCL1/KC production, resulting in diminished cellular infiltration and fungal clearance compared with control mice, especially during infection with clinical isolates expressing high β-glucan. In contrast to Dectin 1^−/− mice, cellular infiltration into infected TLR2^−/−, TLR4^−/−, and MD-2^−/− mice corneas was unimpaired, indicating no role for these receptors in cell recruitment; however, fungal killing was significantly reduced in TLR4^−/− mice, but not TLR2^−/− or MD-2^−/− mice. We also found that TRIF^−/− and TIRAP^−/− mice exhibited no fungal-killing defects, but that MyD88^−/− and IL-1R1^−/− mice were unable to regulate fungal growth. In conclusion, these data are consistent with a model in which β-glucan on A.fumigatus germinating conidia activates Dectin-1 on corneal macrophages to produce IL-1β, and CXCL1, which together with IL-1R1/MyD88-dependent activation, results in recruitment of neutrophils to the corneal stroma and TLR4-dependent fungal killing.     

Corneal Wound Healing Requires IKB kinase β Signaling in Keratocytes

IkB kinase β (IKKβ) is a key signaling kinase for inflammatory responses, but it also plays diverse cell type-specific roles that are not yet fully understood. Here we investigated the role of IKKβ in the cornea using Ikkβ^ΔCS mice in which the Ikkβ gene was specifically deleted in the corneal stromal keratocytes. The Ikkβ^ΔCS corneas had normal morphology, transparency and thickness; however, they did not heal well from mild alkali burn injury. In contrast to the Ikkβ^F/F corneas that restored transparency in 2 weeks after injury, over 50% of the Ikkβ^ΔCS corneas failed to fully recover. They instead developed recurrent haze with increased stromal thickness, severe inflammation and apoptosis. This pathogenesis correlated with sustained myofibroblast transformation with increased α smooth muscle actin (α-SMA) expression, higher levels of senescence β-Gal activity and scar tissue formation at the late stage of wound healing. In addition, the Ikkβ^ΔCS corneas displayed elevated expression of hemo-oxygenase-1 (HO-1), a marker of oxidative stress, and activation of stress signaling pathways with increased JNK, c-Jun and SMAD2/3 phosphorylation. These data suggest that IKKβ in keratocytes is required to repress oxidative stress and attenuate fibrogenesis and senescence in corneal wound healing.     

Analysis of presteady-state Na+ fluxes across the rabbit corneal endothelium

Summary Instead of the conventional steady-state fluxes, the presteady-state fluxes of²²Na across the rabbit corneal endothelium were measured. In contrast to reports that there is no net Na⁺ movement across the corneal endothelium, we find a net transport of Na⁺ across this tissue. The direction of net Na⁺ flux is from the stromal to the aqueous side and the magnitude is 2.3±0.4 eq/cm²·hr (n=11,sem). Net Na⁺ transport is inhibited in the presence of ouabain (10^–4 m). Acetazolamide (10^–4 m) has only a slight inhibitory effect on the rate of Na⁺ transport but decreases the transendothelial potential difference by about 30%. The passive component of the Na⁺ transport has been estimated by analyzing the presteady-state influx and efflux curves and found to occur 10% via cellular and 90% via paracellular routes. The analysis for the separation of the pathways has been based on a recently proposed theory which holds that the flux ratio, regardless of its driving forces, is independent of time.     

The Effect of Riboflavin/UVA Collagen Cross-linking Therapy on the Structure and Hydrodynamic Behaviour of the Ungulate and Rabbit Corneal Stroma

Purpose

To examine the effect of riboflavin/UVA corneal crosslinking on stromal ultrastructure and hydrodynamic behaviour.

Methods

One hundred and seventeen enucleated ungulate eyes (112 pig and 5 sheep) and 3 pairs of rabbit eyes, with corneal epithelium removed, were divided into four treatment groups: Group 1 (28 pig, 2 sheep and 3 rabbits) were untreated; Group 2 (24 pig) were exposed to UVA light (3.04 mW/cm²) for 30 minutes and Group 3 (29 pig) and Group 4 (31 pig, 3 sheep and 3 rabbits) had riboflavin eye drops applied to the corneal surface every 5 minutes for 35 minutes. Five minutes after the initial riboflavin instillation, the corneas in Group 4 experienced a 30 minute exposure to UVA light (3.04 mW/cm²). X-ray scattering was used to obtain measurements of collagen interfibrillar spacing, spatial order, fibril diameter, D-periodicity and intermolecular spacing throughout the whole tissue thickness and as a function of tissue depth in the treated and untreated corneas. The effect of each treatment on the hydrodynamic behaviour of the cornea (its ability to swell in saline solution) and its resistance to enzymatic digestion were assessed using in vitro laboratory techniques.

Results

Corneal thickness decreased significantly following riboflavin application (p<0.01) and also to a lesser extent after UVA exposure (p<0.05). With the exception of the spatial order factor, which was higher in Group 4 than Group 1 (p<0.01), all other measured collagen parameters were unaltered by cross-linking, even within the most anterior 300 microns of the cornea. The cross-linking treatment had no effect on the hydrodynamic behaviour of the cornea but did cause a significant increase in its resistance to enzymatic digestion.

Conclusions

It seems likely that cross-links formed during riboflavin/UVA therapy occur predominantly at the collagen fibril surface and in the protein network surrounding the collagen.     

Geometrical Custom Modeling of Human Cornea In Vivo and Its Use for the Diagnosis of Corneal Ectasia

Aim

To establish a new procedure for 3D geometric reconstruction of the human cornea to obtain a solid model that represents a personalized and in vivo morphology of both the anterior and posterior corneal surfaces. This model is later analyzed to obtain geometric variables enabling the characterization of the corneal geometry and establishing a new clinical diagnostic criterion in order to distinguish between healthy corneas and corneas with keratoconus.

Method

The method for the geometric reconstruction of the cornea consists of the following steps: capture and preprocessing of the spatial point clouds provided by the Sirius topographer that represent both anterior and posterior corneal surfaces, reconstruction of the corneal geometric surfaces and generation of the solid model. Later, geometric variables are extracted from the model obtained and statistically analyzed to detect deformations of the cornea.

Results

The variables that achieved the best results in the diagnosis of keratoconus were anterior corneal surface area (ROC area: 0.847, p<0.000, std. error: 0.038, 95% CI: 0.777 to 0.925), posterior corneal surface area (ROC area: 0.807, p<0.000, std. error: 0.042, 95% CI: 0,726 to 0,889), anterior apex deviation (ROC area: 0.735, p<0.000, std. error: 0.053, 95% CI: 0.630 to 0.840) and posterior apex deviation (ROC area: 0.891, p<0.000, std. error: 0.039, 95% CI: 0.8146 to 0.9672).

Conclusion

Geometric modeling enables accurate characterization of the human cornea. Also, from a clinical point of view, the procedure described has established a new approach for the study of eye-related diseases.     

Contributions of Ocular Surface Components to Matrix-Metalloproteinases (MMP)-2 and MMP-9 in Feline Tears following Corneal Epithelial Wounding

Purpose

This study investigated ocular surface components that contribute to matrix-metalloproteinase (MMP)-2 and MMP-9 found in tears following corneal epithelial wounding.

Methods

Laboratory short-haired cats underwent corneal epithelial debridement in one randomly chosen eye (n = 18). Eye-flush tears were collected at baseline and during various healing stages. Procedural control eyes (identical experimental protocol as wounded eyes except for wounding, n = 5) served as controls for tear analysis. MMP activity was analyzed in tears using gelatin zymography. MMP staining patterns were evaluated in ocular tissues using immunohistochemistry and used to determine MMP expression sites responsible for tear-derived MMPs.

Results

The proMMP-2 and proMMP-9 activity in tears was highest in wounded and procedural control eyes during epithelial migration (8 to 36 hours post-wounding). Wounded eyes showed significantly higher proMMP-9 in tears only during and after epithelial restratification (day 3 to 4 and day 7 to 28 post-wounding, respectively) as compared to procedural controls (p<0.05). Tears from wounded and procedural control eyes showed no statistical differences for pro-MMP-2 and MMP-9 (p>0.05). Immunohistochemistry showed increased MMP-2 and MMP-9 expression in the cornea during epithelial migration and wound closure. The conjunctival epithelium exhibited highest levels of both MMPs during wound closure, while MMP-9 expression was reduced in conjunctival goblet cells during corneal epithelial migration followed by complete absence of the cells during wound closure. The immunostaining for both MMPs was elevated in the lacrimal gland during corneal healing, with little/no change in the meibomian glands. Conjunctival-associated lymphoid tissue (CALT) showed weak MMP-2 and intense MMP-9 staining.

Conclusions

Following wounding, migrating corneal epithelium contributed little to the observed MMP levels in tears. The major sources assessed in the present study for tear-derived MMP-2 and MMP-9 following corneal wounding are the lacrimal gland and CALT. Other sources included stromal keratocytes and conjunctiva with goblet cells.     

The nature of the neutral Na+−Cl− coupled entry at the apical membrane of rabbit gallbladder epithelium: III. Analysis of transports on membrane vesicles

Summary In rabbit gallbladder epithelium, a Na⁺/H⁺, Cl^–/HCO ₃ ^– double exchange and a Na⁺–Cl^– symport are both present, but experiments on intact tissue cannot resolve whether the two transport systems operate simultaneously. Thus, isolated apical plasma membrane vesicles were prepared. After preloading with Na⁺, injection into a sodium-free medium caused a stable intravesicular acidification (monitored with the acridine orange fluorescence quenching method) that was reversed by Na⁺ addition to the external solution. Although to a lesser extent, acidification took place also in experiments with an electric potential difference (PD) equal to 0. If a preset pH difference (pH) was imposed ([H⁺]_in>[H⁺]_out, PD=0), the addition of Na-gluconate to the external solution caused pH dissipation at a rate that followed saturation kinetics. Amiloride (10^–4 m) reduced the pH dissipation rate. Taken together, these data indicate the presence of Na⁺ and H⁺ conductances in addition to an amiloride-sensitive, electroneutral Na⁺/H⁺ exchange.An inwardly directed [Cl^–] gradient (PD=0) did not induce intravesicular acidification. Therefore, in this preparation, there was no evidence for the presence of a Cl^–/OH^– exchange.When both [Na⁺] and [Cl^–] gradients (outwardly directed, PD=0) were present, fluorescence quenching reached a maximum 20–30 sec after vesicle injection and then quickly decreased. The decrease was not observed in the presence of a [Na⁺] gradient alone or the same [Na⁺] gradient with Cl^– at equal concentrations at both sides. Similarly, the decrease was abolished in the presence of both Na⁺ and Cl^– concentration gradients and hydrochlorothiazide (5×10^–4 m). The decrease was not influenced by an inhibitor of Cl^–/OH^– exchange (10^–4 m furosemide) or of Na⁺–K⁺–2Cl^– symport (10^–5 m bumetanide).We conclude that a Na⁺/H⁺ exchange and a Na⁺–Cl^– symport are present and act simultaneously. This suggests that in intact tissue the Na⁺–Cl^– symport is also likely to work in parallel with the Na⁺/H⁺ exchange and does not represent an induced homeostatic reaction of the epithelium when Na⁺/H⁺ exchange is inhibited.     

Typing of hepatic nonparenchymal cells using fibulin-2 and cytoglobin/STAP as liver fibrogenesis-related markers

Fibulin-2 and cytoglobin/stellate cell activation-associated protein (Cygb/STAP) are considered to be markers of hepatic myofibroblasts (MFs) and stellate cells (HSCs), respectively. The aim of the present study was to characterize the nonparenchymal cells (NPCs) of normal rat livers and carbon tetrachloride-induced fibrotic rat livers with respect to the expression of these two proteins. NPCs in normal (Glissons capsules) and fibrotic (fibrotic septa) connective tissues were immunohistochemically categorized into four cell types in terms of the expression of fibulin-2 and Cygb/STAP: fibulin-2 and Cygb/STAP double-positive (Fib⁺/STAP⁺); fibulin-2-positive and Cygb/STAP-negative (Fib⁺/STAP^–); Fib^–/STAP⁺; and Fib^–/STAP^–. The Glissons capsules had Fib⁺/STAP⁺ and Fib^–/STAP^– cell occupancy rates of 45.5% and 54.5%, respectively, but did not contain Fib⁺/STAP^– or Fib^–/STAP⁺ cells. On the other hand, the fibrotic septa contained Fib⁺/STAP⁺, Fib^–/STAP⁺, and Fib^–/STAP^– cells at occupancy rates of 35.0%, 50.5%, and 9.1%, respectively, but did not contain Fib⁺/STAP^– cells. Thus, fibrosis is characterized by a dramatic increase in Fib^–/STAP⁺ NPCs, and a dramatic decrease in Fib^–/STAP^– NPCs. Fib⁺/STAP⁺ NPCs are located uniformly in Glissons capsules and peripherally in fibrotic septa. The present study strongly suggests that Fib⁺/STAP⁺ and Fib^–/STAP⁺ NPCs correspond to MFs and activated HSCs, respectively, both of which may contribute to liver fibrogenesis.     

Cellular and paracellular pathway resistances in the “tight” Cl−-secreting epithelium of rabbit cornea

Summary The high transverse resistance of the isolated rabbit cornea (6–12 k·cm²) is associated with the corneal epithelium, a Cl^–-secreting tissue which is modulated by -adrenergic and serotonergic receptors. Three methods were employed to determine the resistances for the apical membrane, basolateral membrane, and paracellular conductive pathways in the epithelium. In the first method, the specific resistance of the apical membrane was selectively and reversibly changed. Epinephrine was used to increase apical Cl^– conductance and Ag⁺ was used to increase apical cation permeability. The second method utilized a direct measure of the spontaneous cellular ionic current. The third method obtained estimates of shunt resistance using transepithelial electrophysiological responses to changes in apical membrane resistance. The results of the first method were largely independent of the agent used. In addition, the three methods were in general agreement, and the ranges of mean values for apical membrane, basolateral membrane, and shunt resistances were 23–33, 3–4, and 12–16 k·cm², respectively, for the normal cornea. The apical membrane was the major, physiologically-modulated barrier to ion permeation. The shunt resistance of the corneal epithelium was comparable to that found previously for other tight epithelia. Experiments using Ag⁺ in tissues that were bathed in Cl^– and HCO₃-free solutions indicated that under resting conditions the apical membrane is anion-selective.