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J V Jester

Publications and source records attributed to J V Jester.

At least 55 records · Page 3Linked to original sources
PubMed1998

Neutralizing antibody to TGFbeta modulates stromal fibrosis but not regression of photoablative effect following PRK.

PURPOSE: Previous studies have suggested that corneal fibrosis controlled by a TGFbeta-mediated cytocrine pathway underlies the development of clinical corneal haze and associated regression of photoablative effect following excimer laser PRK. Using a unique blocking antibody, we evaluated the role of TGFbeta in post-PRK corneal wound healing as measured by in vivo Confocal Microscopy Through Focusing (CMTF). METHODS: Twelve rabbits received a monocular, 6-mm diameter, 9.0 D PRK myopic correction. Six animals received 50 microg of anti-TGFbeta blocking antibodies applied topically 3x/day for three days post-PRK, while six animals received vehicle alone. An additional six animals served as unoperated controls. At various times during a four-month-period, animals were evaluated using CMTF, which generates a quantitative image intensity depth profile of the cornea. The location and reflectivity of corneal structures were identified from CMTF-profiles and used to determine epithelial and stromal thickness and corneal light reflectivity as an objective estimate of corneal haze. To correlate in vivo and ex vivo morphology, an additional six rabbits were analyzed at differing temporal intervals post-PRK for the expression and cytoskeletal organization of contractile microfilaments: f-actin (stress fibers) and alpha-smooth muscle actin (a molecular marker for myofibroblast transformation). RESULTS: Anti-TGFbeta treated corneas showed significantly less CMTF measured light reflectivity (ANOVA, p < 0.02) following PRK compared to vehicle treated corneas with a 34% decrease at two weeks (2513 +/- 758 U compared to 3810 +/- 1262 U) and a 61% reduction in reflectivity at four months (447 +/- 208 U compared to 1154 +/- 585 U). The reduction in early development of light reflecting structures and the more rapid decline appeared related to anti-TGFbeta-mediated inhibition of keratocyte activation and proliferation, myofibroblast transformation, and stromal fibrosis. Between anti-TGFbeta and vehicle treated corneas, no significant differences were detected in either photoablation depth (126 +/- 9 microm versus 126 +/- 7 microm) or regression of photoablative effect (postoperative stromal thickening at four months: 95 +/- 16 microm versus 95 +/- 10 microm). Histologic examination demonstrated that regression of photoablative effect in anti-TGFbeta treated corneas was related entirely to regeneration by corneal growth underlying the photoablated stromal surface. In vehicle treated corneas, fibrosis or deposition of new fibrotic tissue above the photoablated stromal surface was observed but contributed only about 25% of the total postoperative stromal thickening. No epithelial hyperplasia was detected. In unoperated control animals, a physiologic stromal thickening of 5 +/- 2 microm per month (p < 0.001) was observed. CONCLUSIONS: This study confirms our earlier observations that increased corneal light reflectivity following PRK is predominantly due to: (1) distortion of the photoablated stromal surface leading to prominent reflections; and (2) increased reflections from activated and transformed keratocytes. Anti-TGFbeta reduced keratocyte activation and transformation and inhibited stromal fibrosis, leading to a reduction in early light reflectivity as well as to a more rapid decline. Of greatest interest is the unexpected finding that anti-TGFbeta treatment inhibited stromal fibrosis without reducing or delaying post-PRK stromal re-thickening. Based on these findings we propose that corneal thickness may be tightly and dynamically regulated by an unknown, non-TGFbeta mediated pathway. We propose that anti-TGFbeta treatment may be useful in reducing post-PRK corneal haze development in patients by: (1) inhibiting the recruitment of highly reflective, activated keratocytes, (2) inhibiting myofibroblast transformation, and 3) reducing stromal fibrosis.

Actins↗
PubMed1998

Myofibroblast transformation of cat corneal endothelium by transforming growth factor-beta1, -beta2, and -beta3.

PURPOSE: Under certain pathophysiologic conditions, the corneal endothelium can produce an abnormal posterior collagenous layer (PCL) that reduces light transmission. Previous studies suggest that formation of PCLs can result from transformation of endothelial cells to a proliferative myofibroblast phenotype. The purpose of this study was to determine the potential role of transforming growth factor (TGF)-beta on corneal endothelial transformation. METHODS: Three corneal buttons (6-mm diameter) were obtained from each cornea of 28 adult cats. After a 2-mm diameter mechanical scrape injury was made, each button was cultured for 24, 48, or 72 hours in serum-free medium (SFM) or SFM supplemented with 10% fetal calf serum, TGF-gamma1, TGF-beta2, TGF-beta3, basic fibroblast growth factor (bFGF), or TGF-beta1 and bFGF. Buttons were single and double labeled using phalloidin and antibodies to ZO-1, Ki67, fibronectin, alpha-smooth muscle (SM) actin, and vinculin. Counts of Ki67-positive cells were used as a measure of endothelial proliferation. RESULTS: Organ culture in TGF-beta1, beta2, or beta3 induced myofibroblast transformation of corneal endothelial cells, with formation of stress fibers containing alpha-SM actin, loss of normal pericellular ZO-1 organization, development of extracellular fibronectin fibrils, and formation of focal contacts as indicated by punctate vinculin staining. However, TGF-beta3 did not stimulate endothelial proliferation above that in serum-free control samples. Serum and bFGF each stimulated proliferation significantly, without inducing myofibroblast transformation. A combination of TGF-beta1 and bFGF resulted in both myofibroblast transformation and increased proliferation. CONCLUSIONS: These results suggest that TGF-beta plays a key role in the loss of normal endothelial differentiation, abnormal extracellular matrix synthesis, and myofibroblast transformation, which can induce development of PCLs. However, other factors such as bFGF seem to be required to stimulate concomitant proliferation of corneal endothelium.

Animals↗
PubMed1998

Area and depth of surfactant-induced corneal injury predicts extent of subsequent ocular responses.

PURPOSE: To correlate area and depth of initial corneal injury induced by surfactants of differing type and irritant properties with corneal responses and outcome in the same animals over time by using in vivo confocal microscopy (CM). METHODS: Six groups of six adult rabbits were treated with anionic, cationic, and nonionic surfactants that caused different levels of ocular irritation. Test materials included slight irritants: 5% sodium lauryl sulfate (SLS), polyoxyethylene glycol monoalkyl ether (POE), and 5% 3-isotridecyloxypropyl-bis(polyoxyethylene) ammonium chloride (ITDOP); mild irritants: 5% 3-decyloxypropyl-bis(polyoxyethylene) amine (DOP) and sodium linear alkylbenzene sulfonate (LAS); and a moderate irritant: a proprietary detergent (DTRGT). Ten microliters surfactant were directly applied to the cornea of one eye of each rabbit. Ten untreated rabbits served as control subjects. Area and depth of initial injury was determined by using in vivo CM to measure epithelial thickness, epithelial cell size, corneal thickness, and depth of stromal injury in four corneal regions at 3 hours and at day 1. Area and depth of corneal responses to injury were evaluated at various times from days 3 through 35 by macroscopic grading and quantitative confocal microscopy through-focusing (CMTF). RESULTS: In vivo CM revealed corneal injury with slight irritants to be restricted to the epithelium, whereas the mild and moderate irritants caused complete epithelial cell loss with increasing anterior stromal damage: DOP < LAS < DTRGT. With the slight ocular irritants there was little or no change in corneal thickness or the CMTF intensity profiles. Three hours after treatment, mild and moderate ocular irritants caused a significant increase in corneal thickness, which peaked at day 1 with DOP (483.3+/-80.1 microm) and LAS (572.3+/-60.0 microm) and day 3 with DTRGT (601.4+/-68.7 microm); returning to normal (similar to control values) by day 7 with DOP and day 35 with LAS and DTRGT. The CMTF intensity profiles also showed significant elevation over that in the anterior stroma, which peaked at day 1 with DOP (14,608+/-4,306 U [U is defined as micrometers X pixel intensity]) and day 3 with LAS and DTRGT (18,471+/-6,581 U and 22,424+/-3,704 U, respectively) and returned toward normal by day 7 with DOP and day 14 with LAS and DTRGT. Elevated CMTF profiles principally reflected the presence of hyperreflective, punctate keratocytes and inflammatory cells at days 1 and 3 and the presence of activated keratocytes at day 7. There was a significant correlation between the elevated CMTF intensity profile and the corresponding macroscopic total score in each eye (r = 0.839; P < 0.001). More important, there was a significant correlation between area and depth of initial stromal injury measured at day 1, regardless of ocular irritant and the stromal response measured by the area under the CMTF intensity profile curve in each cornea (r = 0.87; P < 0.0005). A significant correlation between the area and depth of injury and the area under the corneal thickness curve was also observed in each cornea (r = 0.75; P < 0.0005). CONCLUSIONS: In individual animals, the extent of initial stromal injury correlated with the magnitude of the corneal responses, measured by the change in corneal thickness and the CMTF depth intensity profile. These findings further support the hypothesis that area and depth of injury are the principal factors determining the early responses and eventual repair processes after accidental eye irritation. They also support the proposed use of area and depth of acute injury as a mechanistic correlate to ocular irritation in the development and validation of potential in vitro ocular irritation tests.

Animals↗
PubMed1997

An in vitro force measurement assay to study the early mechanical interaction between corneal fibroblasts and collagen matrix.

An in vitro force measurement assay has been developed to quantify the forces exerted by single corneal fibroblasts during the early interaction with a collagen matrix. Corneal fibroblasts were sparsely seeded on top of collagen matrices whose stiffness was predetermined by micromanipulation with calibrated fine glass microneedles. The forces exerted by individual cells were calculated from time-lapse videomicroscopic recordings of the 2-D elastic distortion of the matrix. In additional experiments, the degree of permanent reorganization of the collagen matrices was assessed by lysing the cells with 1% Triton X-100 solution at the end of a 2-hour incubation and recording the subsequent relaxation. The data suggest that a cell can exert comparable centripetal force during either extension of a cell process or partial retraction of an extended pseudopodia. The rates of force associated with pseudopodial extension and partial retraction were 0.180 +/- 0.091 (x 10(-8)) N/min (n = 8 experiments) and 0.213 +/- 0.063 (x 10(-8)) N/min (n = 8 experiments), respectively. Rupture of pseudopodial adhesion associated with cell locomotion causes a release of force on the matrix and a complete recoil of the pseudopodia concerned; a simultaneous release of force on the matrix was also observed at the opposite end of the cell. Lysis of cells resulted in 84 +/- 18% relaxation of the matrix, suggesting that little permanent remodeling of matrix is produced by the actions of isolated migrating cells.

Animals↗
PubMed1997

ZO-1 reorganization and myofibroblast transformation of corneal endothelial cells after freeze injury in the cat.

Corneal endothelial wound healing following scrape injury in the rabbit and cat is characterized by cell spreading and maintenance of a normal endothelial phenotype consisting of apically-localized, circumferential microfilament bands and cell border-associated ZO-1, a tight junction protein and marker for endothelial differentiation. In contrast, after freeze injury in the rat and rabbit endothelial cells develop basally organized microfilament bundles (stress fibers), and appear to proliferate and form a multilayered zone at the wound margin. The purpose of the present study was to determine if similar phenotypic changes are observed after freeze injury in the cat corneal endothelium, which like human, normally has limited growth potential. In addition, changes in ZO-1 and alpha-smooth muscle actin (a marker for myofibroblast transformation) distribution were evaluated for the first time following freeze injury. In vivo endothelial healing of standard 3 mm diameter freeze injury was evaluated at 4 hr, 12 hr, 24 hr, 48 hr, 3 days and 5 days after injury in 22 cat eyes. Corneas were stained with phalloidin, propidium iodide, and anti-ZO-1, anti-alpha-smooth muscle-specific actin or anti-fibronectin antibodies. Protein organization was then evaluated using immunofluorescence and laser scanning confocal microscopy. Beginning at 12 hr after injury, endothelial cells appeared to extend and elongate over the wound area. By 48 hr after injury, migrating endothelial cells formed a multilayered activated zone (AZ) at the wound margin. Endothelial cells immediately adjacent to the AZ maintained a normal circumferential organization of f-actin colocalized with cell border-associated anti-ZO-1 staining at all time points observed. However, within the AZ there was an abrupt increase in phalloidin staining and development of prominent microfilament bundles (stress fibers), as well as a loss of normal anti-ZO-1 staining. The AZ also stained positively for anti-alpha-smooth muscle actin and anti-fibronectin antibodies. Changes in the distribution of ZO-1 were observed as early as 4 hr after injury, and appeared to precede f-actin reorganization. These data indicate that endothelial healing after freeze injury in the cat involves a loss of normal endothelial differentiation and cell connectivity, and transformation to a myofibroblastic phenotype.

Actin Cytoskeleton↗
PubMed1997

Confocal microscopic characterization of initial corneal changes of surfactant-induced eye irritation in the rabbit.

We have previously demonstrated with slightly and severely irritating surfactants that the new technology of noninvasive, in vivo confocal microscopy (CM) can be a useful approach to a better understanding of the pathobiology of ocular irritation in situ. In this study, in vivo CM was used to qualitatively and quantitatively characterize the initial microscopic corneal changes occurring with surfactants of slight, mild, moderate, and severe irritation. Surfactants were directly applied to the corneas of rabbits (6/group) at a dose of 10 microl. Eyes and eyelids were examined macroscopically and scored for irritation beginning at 3 hr after dosing and periodically through Day 35. Concurrently, the corneas were evaluated by in vivo CM; 3D data sets extending from the surface epithelium to the endothelium were assessed for surface epithelial cell size, epithelial layer thickness, total corneal thickness, and depth of keratocyte necrosis. The average macroscopic scores at 3 hr for the slight, mild, moderate, and severe irritants were 6.0, 39.3, 48.5, and 68.7, respectively, of a possible 110. At 3 hr, in vivo CM revealed corneal injury with the slight irritant limited to the epithelium, resulting in reductions in epithelial cell size and thickness to 59.0 and 82.4% of controls (p < 0.001 and p < 0.01, respectively). These parameters returned to normal by Day 3. For the mild irritant, at 3 hr the epithelium was absent, corneal thickness was increased to 157.6% of controls (p < 0.001), and necrosis of keratocytes extended to an average depth of 4.3 microm (0.8% of the corneal thickness); these parameters were essentially normal by Day 14. For the moderate irritant, at 3 hr the epithelium was markedly attenuated, corneal thickness was increased to 155.8% of controls (p < 0.001), and keratocyte necrosis extended to an average depth of 19.0 microm (3.6% of corneal thickness; statistically greater than with the mild irritant, p < 0.001); these parameters were essentially normal by Day 14. For the severe irritant, at 3 hr the epithelium was significantly thinned, corneal thickness was increased to 165.9% of controls (p < 0.001), and keratocyte necrosis occurred to an average depth of 391.1 microm (70.1% of corneal thickness). These findings demonstrate that significant differences in area and depth of injury occur with surfactants of differing irritancy. The data suggest that differences at 3 hr can be used to distinguish different levels of ocular irritation. Data such as these will be important in the development and evaluation of future mechanistically based in vitro alternatives for ocular irritancy testing.

Animals↗
PubMed1997

Quantification of stromal thinning, epithelial thickness, and corneal haze after photorefractive keratectomy using in vivo confocal microscopy.

PURPOSE: The authors establish, for the first time, observer-independent quantification of stromal thinning, epithelial thickness, and corneal haze after excimer laser photorefractive keratectomy (PRK) using a unique, new form of in vivo confocal microscopy. METHODS: Rapid, continuous z-scans of high-resolution confocal images, termed confocal microscopy through focusing (CMTF), were performed in the central corneal area of 17 patients before and 1 month after PRK for low- to moderate-grade myopia (-2.88-9.13 diopters [D]). Corneal, epithelial, and stromal thickness measurements and an objective haze estimate were obtained from each CMTF scan by digital image analysis. RESULTS: Epithelial thickness averaged 51 +/- 4 microns before and 45 +/- 10 microns 1 month post-PRK (P < 0.005), whereas stromal thinning ranged from 20 to 154 microns, representing a direct estimate of the actual photoablation depth. Corneal thickness averaged 560 +/- 36 microns before PRK and 462 +/- 52 microns at 1 month. The change in corneal thickness correlated closely with the change in spherical equivalent refraction (r = 0.94, P < 0.0001); linear regression analysis revealed a value of 14.3 microns corneal thinning per diopter of correction. A significant correlation was found between the objective CMTF haze estimate and a clinical haze grading obtained by slit-lamp examination (r = 0.73, P < 0.001). CONCLUSIONS: Confocal microscopy through focusing is a new, powerful in vivo tool that enables quantitative, unbiased evaluation of PRK procedures over time by providing epithelial and stromal thickness analysis, photoablation depth assessment, and unbiased haze measurement. The method is uniquely valuable in the pre- and postoperative assessment of PRK patients and for determining the optimal treatment strategy, especially in assessing refractive and visual outcomes in individual cases.

Adult↗
PubMed1997

Epithelial and corneal thickness measurements by in vivo confocal microscopy through focusing (CMTF).

PURPOSE: To study the feasibility of measuring total corneal thickness, as well as the thickness of the epithelium and Bowman's layer, using a novel in vivo confocal microscopy through-focusing (CMTF) methodology. METHODS: The central cornea was scanned from the epithelium to endothelium at an average focal plane speed of 32 microns/sec for rabbits, and 64 microns/sec for humans. Scans were initially video-recorded and later digitized. From digital images, CMTF intensity curves were generated by calculating the average pixel intensity in the central 180 x 180 pixel region (285 microns x 285 microns) of each image in the scan, and plotting as a function of z-depth. Peaks in this intensity profile were then empirically correlated to unique corneal layers using a program which interactively displayed images corresponding to the mouse cursor position along the intensity profile curve. Sublayer thickness values were then calculated from the z-axis positions of the relevant peaks in the intensity curve. Ten normal rabbits and seven human volunteers were evaluated in the study. Both CMTF and ultrasonic pachymetry (UP) measurements were performed on rabbit eyes to determine the agreement between CMTF and UP. RESULTS: Distinct epithelial, basal lamina, and endothelial peaks were identified for all 10 rabbit eyes. The mean central corneal thickness in the rabbit was 381.6 +/- 27.3 microns by CMTF and 384.4 +/- 28.7 microns by UP. The mean difference in central corneal thickness between CMTF and UP was -2.8 +/- 7.1 microns which was not statistically significant (p > 0.2 by paired t-test). Central epithelial thickness in the rabbit measured by CMTF was 47.7 +/- 2.2 microns. The average coefficients of variation for repeated scans were 2.5% and 0.7% for epithelial and corneal thickness, respectively. The standard errors for both epithelial and corneal thickness were less than 1.5 microns for all rabbits. The reproducibilities for epithelial and corneal thickness measurements were 2.2 microns and 2.6 microns, respectively, calculated as the square root of the within group variances of One-Way ANOVA. Intensity profiles for human corneas showed strong epithelial and endothelial peaks, as well as smaller peaks corresponding to the basal-epithelial nerve plexus and the denser anterior layer of stromal keratocyte nuclei. The mean central corneal thickness in the human was 532.1 +/- 18.8 microns; central epithelial thickness was 50.6 +/- 3.9 microns; central Bowman's layer thickness was 16.6 +/- 1.1 microns. The average coefficients of variation for repeated scans were 5.9%, 13.2%, and 1.6% for epithelial, Bowman's layer, and corneal thickness, respectively. The standard errors for all measurements were less than 2.4 microns. The reproducibilities for epithelial, Bowman's layer, and corneal thickness measurements were 3.2 microns, 2.3 microns, and 10.0 microns, respectively. CONCLUSIONS: CMTF is a novel, reproducible technique for obtaining epithelial and corneal thickness measurements during clinical in vivo confocal microscopy of the cornea. More importantly, this methodology provides the first objective, quantitative approach for measurement and analysis of depth and thickness of corneal sub-layers which may prove uniquely valuable in temporally assessing corneal function.

Adult↗
PubMed1997

Characterization of SV40-transfected cell strains from rabbit keratocytes.

The process of corneal wound healing involves the transformation of adjacent corneal keratocytes to myofibroblast-like cells characterized by the development of prominent microfilament bundles containing alpha-smooth muscle-specific actin (alpha-SM), a contractile protein thought to be important in mediating wound contraction. Recent studies have shown that the expression of alpha-SM in cultured corneal keratocytes can be induced by serum and TGF beta 1. To study the cellular and molecular mechanisms underlying this transformation process and to begin to identify the role of alpha-SM in wound contractile events, we generated immortalized rabbit corneal cell strains with extended life by using SV40 transfection. Two unique strains were isolated (TRK-36 and TRK-43). TRK-36, which appears similar to normal corneal keratocytes, maintains a stellate, keratocyte morphology when grown in the absence of serum and transforms to a myofibroblast-like cell when treated with TGF beta 1 (1 ng/ml), as indicated by the induced expression of alpha-SM actin. TRK-43 exhibits features characteristic of myofibroblasts in that it constitutively expresses alpha-SM actin under serum-free conditions. Both strains show in vitro contraction of collagen gels < or = 80% in 24 h in serum-containing medium. Interestingly, under serum-free conditions, TRK-43 cells showed significantly greater contraction of collagen gels compared with those of TRK-36. Overall, the establishment and further study of these cell strains may provide important insights into the molecular mechanisms underlying myofibroblast transformation.

Actins↗
PubMed1997

Adherence of Pseudomonas aeruginosa to shed rabbit corneal epithelial cells after overnight wear of contact lenses.

PURPOSE: Previous studies have shown that contact lens oxygen transmissibility correlates with binding of Pseudomonas aeruginosa to the rabbit cornea after overnight lens wear. Studies of human lens wear stratified by oxygen transmissibility will be required to validate these animal results. In humans, bacterial binding to shed cells obtained through corneal irrigation cytology may provide an indirect measure of in vitro binding. The purpose of this study was to establish the relationship between binding to shed cells and to the residual corneal surface in an animal model of lens wear prior to initiation of human studies. METHODS: The test contact lenses used were: rigid lens A (Dk/L = 10 x 10(-9) [cm/ sec][mL O2/mL mmHg]); rigid lens B (Dk/L = 97); soft lens A (Dk/L = 9); soft lens B (Dk/L = 20); and, soft lens C (Dk/L = 39). There were six rabbits in each group, except for the soft lens C group, which had seven rabbits. After overnight lens wear, the corneal surface was irrigated with a corneal irrigation chamber to collect surface cells before exposure to a bacterial suspension (1 x 10(7) CFU/mL) for 30 minutes. The number of bacteria adherent to the residual corneal surface was then assessed by CFU determination. Cells collected from the corneal surface (9 mL) were incubated with 1 mL bacterial suspension containing 10(8) (CFU/mL) for 30 minutes. The number of bacteria adherent to shed cells was assessed by staining with acridine orange and direct counting by epifluorescence microscopy. RESULTS: The differences in the number of bacteria adhering to shed epithelial cells between the treated and the control eyes were 2.90 +/- 1.20 and 0.23 +/- 0.41 for rigid lenses A and B, respectively, and 5.97 +/- 1.54, 3.67 +/- 2.32, and 0.90 +/- 1.45 (bacterial/cell) for soft lenses A, B, and C, respectively. Overnight contact lens wear induced a significant increase in bacterial binding to shed corneal epithelial cells for rigid lens A and for soft lenses A and B. There were significant differences among lens groups (P = 0.00017, ANOVA), with significant differences between rigid lenses A and B, soft lenses A and C, and soft lenses B and C. The binding of bacteria to shed cells was significantly correlated with the binding of bacteria to the residual corneal surface, both confirming and extending previous results (R = 0.78, P < 0.001). CONCLUSION: These results demonstrate a positive correlation between P. aeruginosa adherence to shed corneal cells and to the residual corneal surface in the rabbit eye following contact lens wear. In light of the results from prior animal studies, examination of the behavior of P. aeruginosa binding to exfoliated cells appears to be a promising and valid method for future assessment of similar lens-induced increases in bacterial binding in prospective human clinical studies.

Animals↗
PubMed1997

Inhibition of corneal fibrosis by topical application of blocking antibodies to TGF beta in the rabbit.

Previous studies have shown that TGF beta 1 induces activation and myofibroblast transformation of cultured rabbit corneal keratocytes. To determine whether TGF beta has a similar function in vivo, we evaluated the effect of TGF beta-blocking antibodies on corneal fibrosis after lamellar keratectomy (LK) in the rabbit. A total of 51 rabbits received standard LK wounds, and eyes were treated with 50 microliters of Celluvisc/PBS, containing 10, 50, or 100 micrograms of 1D11, a mouse monoclonal anti-TGF beta-blocking antibody. Control wounds received either 100 micrograms of an irrelevant mouse monoclonal antibody or vehicle alone. At days 14, 28, 42, and 56, eyes were evaluated by in vivo confocal microscopy (CM) and the mice were killed for light microscopy (LM) and immunostaining with antibodies to human fibronectin. In vivo CM of LK wounds clearly identified a disorganized layer that contained irregularly arranged fibroblasts and reflective extracellular matrix overlying normal corneal stroma. In a subset of 11 eyes stained with 5-(4,6-dichlorotriazinyl) aminofluorescein (DTAF) immediately after injury, the thickness of the disorganized layer identified by in vivo CM significantly correlated with both anterior corneal fibrosis (r = 0.627; p < 0.025) and depth of keratocyte activation (r = 0.8980; p < 0.0005), indicating that in vivo CM can be used quantitatively to assess anterior stromal fibrosis. In eyes treated with an irrelevant monoclonal antibody, in vivo corneal fibrosis averaged 100 +/- 26 microns thick at day 14, whereas treatment with 10, 50, and 100 micrograms anti-TGF beta significantly reduced (p < 0.0005) the anterior disorganization in a dose-dependent fashion to 101 +/- 32, 45 +/- 11, and 56 +/- 18 microns, respectively. Semiquantitative measurement of anti-fibronectin staining within the wound revealed that anti-TGF beta significantly reduced the intensity of anti-fibronectin staining in the anterior 50 microns of the corneal stroma (p < 0.003). These findings indicate that TGF beta plays an important in vivo role in keratocyte activation and myofibroblast transformation. Furthermore, the in vivo use of TGF beta-blocking antibody effects may allow modulation of corneal fibrosis after refractive surgery.

Administration, Topical↗
PubMed1996

Quantitative three-dimensional confocal imaging of the cornea in situ and in vivo: system design and calibration.

A new depth encoding system (DES) is presented, which makes it possible to calculate, display, and record the z-axis position continuously during in vivo imaging using tandem scanning confocal microscopy (TSCM). In order to verify the accuracy of the DES for calculating the position of the focal plane in the cornea both in vitro and in vivo, we compared TSCM measurements of corneal thickness to measurements made using an ultrasonic pachymeter (UP, a standard clinical instrument) in both enucleated rabbit, cat, and human eyes (n = 15), and in both human patients (n = 7). Very close agreement was found between the UP and TSCM measurements in enucleated eyes; the mean percent difference was 0.50 +/- 2.58% (mean +/- SD, not significant). A significant correlation (R = 0.995, n = 15, p < 0.01) was found between UP and TSCM measurements. These results verify that the theoretical equation for calculating focal depth provided by the TSCM manufacturer is accurate for corneal imaging. Similarly, close agreement was found between the in vivo UP and TSCM measurements; the mean percent differences was 1.67 +/- 1.38% (not significant), confirming that z-axis drift can be minimized with proper applanation of the objective. These results confirm the accuracy of the DES for imaging of the cornea both ex vivo and in vivo. This system should be of great utility for applications where quantitation of the three-dimensional location of cellular structures is needed.

Adult↗
PubMed1996

Measurement of surgically induced corneal deformations using three-dimensional confocal microscopy.

The goal of this study was to develop and apply a new set of experimental techniques for measuring the local deformations induced by partial-thickness corneal incisions in situ. Eight adult cat eyes were enucleated and cannulated, with corneal viability maintained as close to in vivo conditions as possible and intraocular pressure (IOP) carefully controlled. Experimental measurements were made pre/post radial keratotomy (RK) surgery in situ at IOPs of 15, 30, and 45 mm Hg. Incision depth and cross-sectional profiles were measured at the midpoint of selected incisions using three-dimensional (3-D) tandem scanning confocal microscopy (TSCM); central corneal curvature was estimated using a commercial corneal topographical analysis system, and corneal thickness was assessed by both 3-D TSCM and ultrasonic pachymetry. Corneas were then processed for light microscopy and incision depth was measured histologically. Finite element models were developed for comparison with the experimental measurements. There was no significant change in central corneal thickness (-5.3 +/- 3.9%, n = 8) over the course of the experiments, demonstrating that normal endothelial cell function and normal stromal hydration was maintained. The in situ TSCM incision depth measurements were significantly correlated with the histological measurements (slope = 0.95, R = 0.854, p < 0.01, n = 13 incisions). Measured incision gape at the top (anterior) of the stroma was 64.9 +/- 13.4, 87.3 +/- 12.6, and 108.7 +/- 14 microns at IOPs of 15, 30, and 45 mm Hg, respectively. The 3-D incision profiles were nonlinear in shape; comparison with the finite element models suggests that the shape of the wound profile may provide unique information regarding the shear stiffness of the cornea. Overall, the data suggest that TSCM measurements of the cross-sectional profile of the incisions immediately after RK under controlled in situ conditions provide important data regarding the mechanical behavior of the cornea after refractive surgery. These data should provide the foundation for future studies into the relationships between local tissue mechanics and corneal wound healing.

Animals↗
PubMed1996

The Fas-Fas ligand system and other modulators of apoptosis in the cornea.

PURPOSE: Previous studies have suggested that the disappearance of anterior keratocytes after injury to the overlying epithelium is mediated by apoptosis. The authors examined the expression of the apoptosis-related modulators, Fas (receptor), Fas ligand, Bax, Bcl-2, Bcl-XL, and interleukin-1 beta converting enzyme (ICE) in corneal cells as candidate mediators of this response and tested the effect of Fas receptor-stimulating antibody on corneal stromal fibroblast cells in vitro. METHODS: Reverse-transcription-polymerase chain reaction was used to detect FAS, FAS ligand, Bax, Bcl-2, Bcl-XL, and ICE mRNA expression in primary cultures of human corneal epithelial, stromal fibroblast, and endothelial cells. Immunohistochemistry was applied to detect Fas and Fas ligand proteins in fresh-frozen sections of normal human cornea. The effect of FAS-stimulating monoclonal antibody on first-passage stromal fibroblasts was studied using a DNA fragmentation assay, the live-dead assay with fluorescent microscopy, toluidene blue staining with light microscopy, and electron microscopy. RESULTS: FAS, Fas ligand, Bax, Bcl-2, Bcl-XL, and ICE mRNAs are expressed in all three major cell types of the cornea. Fas protein is expressed in corneal epithelial, keratocyte, and endothelial cells in fresh-frozen human cornea. Fas ligand protein, however, was detected in corneal epithelial and endothelial, but not keratocyte, cells. Fas-stimulating antibody induced first-passage stromal fibroblast cell death with morphologic changes and DNA fragmentation consistent with apoptosis. CONCLUSIONS: The Fas system (Fas and Fas ligand) modulators and final common pathway mediators of apoptosis are expressed in corneal cells. The distribution of Fas (epithelial, keratocyte, and endothelial cells) and Fas ligand (epithelial and endothelial cells) protein expression in fresh-frozen corneal tissue suggests that Fas ligand expressed in corneal epithelial and endothelial cells modulates functions in keratocyte cells and, possibly, autocrine-juxtacrine functions in epithelium and endothelium. The Fas-Fas ligand system is expressed in the cornea and could have important functions in normal corneal physiology and in the pathophysiology of corneal disease, including modulation of keratocyte apoptosis after epithelial injury.

Amino Acid Sequence↗
PubMed1996

Induction of alpha-smooth muscle actin expression and myofibroblast transformation in cultured corneal keratocytes.

The effects of serum, transforming growth factor (TGF) beta 1, bFGF, and heparin on in vitro myofibroblast transformation was studied. Primary rabbit corneal keratocytes were grown under serum-free conditions or in media supplemented with serum (10% fetal calf serum), TGF beta 1 (0.1-10 ng/ml), basic fibroblast growth factor (bFGF) (0.1-10 ng/ml), or heparin (10 U/ml). Cells were analyzed for expression of alpha-smooth muscle actin (alpha-SM actin), alpha 5 beta 1 integrin (the high-affinity fibronectin receptor) and fibronectin by immunoprecipitation, Western blotting, and immunofluorescence. Corneal keratocytes grown in the presence of serum showed a typical fibroblast morphology with induction of alpha-SM actin expression in 1 to 10% of cells. Addition of bFGF blocked serum-induced alpha-SM actin expression, whereas addition of TGF beta 1 enhanced alpha-SM actin expression (100%), which in combination with heparin (10 U/ml), led to a pulling apart of the fibroblastic sheet, simulating contraction. Under serum-free conditions, with or without bFGF and heparin, primary corneal fibroblasts appeared morphologically similar to in situ corneal keratocytes, demonstrating a broad, stellate morphology with interconnected processes and no alpha-SM actin expression. Addition of TGF beta 1 to serum-free cultures resulted in a dramatic transformation of corneal keratocytes to spindle-shaped, fibroblast-like cells that expressed alpha-SM actin in 100% of cells and exhibited a 20-fold increase in fibronectin synthesis and a 13-fold increase in alpha 5 beta 1-integrin synthesis. These effects were blocked by the addition of neutralizing antibodies (16 micrograms/ml). Overall these data suggest that TGF beta 1 is a potent modulator of myofibroblast transformation under serum-free conditions. In addition, the growth of keratocytes in serum appears to mimic, in part, in vivo activation and myofibroblast transformation. We conclude that detailed study of TGF beta 1-induced myofibroblast transformation under defined serum-free conditions will provide important insights into the myofibroblast transformation process.

Actins↗
PubMed1996

Effects of basic FGF and TGF beta 1 on F-actin and ZO-1 organization during cat endothelial wound healing.

Previous studies suggest the existence of two separate and distinct mechanisms of endothelial wound healing (i.e., cell migration and cell spreading), which may be controlled by unique, injury-dependent, wound-related factors. The purpose of our study was to evaluate potential biologic mediators regulating healing of the growth arrested cat endothelium by using an ex vivo, organ-culture model. Three buttons were punched from each cornea of 11 cats with a 6-mm trephine. A 1- to 2-mm diameter endothelial scrape injury (SI) was made, and buttons were cultured in (a) serum-free media (SFM), (b) serum plus media (20% fetal calf serum), (c) SFM plus basic fibroblast growth factor (bFGF), (d) SFM plus bFGF and heparin, (e) SFM plus transforming growth factor-beta 1 (TGF beta 1), or (f) SFM plus TGF beta 1 and anti-TGF beta 1. At various times from 8-48 h after injury, buttons were stained with phalloidin and anti-ZO-1, and imaged by using laser scanning confocal microscopy. Evaluation of SI in cat corneal buttons under serum-free conditions showed maintenance of normal endothelial differentiation, indicating that the organ-culture SI model mimics in vivo SI. Addition of TGF beta 1 produced a dramatic reorganization of apical F-actin and development of stress fibers, as well as the loss of normal cell border-associated ZO-1 distribution. The effects of TGF beta 1 were blocked by the neutralizing antibodies to TGF beta 1. Addition of serum or bFGF produced much less pronounced changes in F-actin and ZO-1 distribution. These results suggest that TGF beta 1 may play a critical role in modulating the wound-healing response of the corneal endothelium.

Actins↗
PubMed1995

Basic science and applications of in vivo microscopy.

Confocal microscopy creates a scanned image from a point light source and point detection or a scanning slit to remove scattered light and improve optical resolution. This also results in optical sectioning of tissues. These capabilities can be employed to image structures in the human cornea, in vivo, both for research and for the diagnosis and treatment of human disease. Optical sections, when recombined, can lead to three-dimensional reconstructions from which very useful information is obtained. Investigators have found keratocyte density decreases from anterior to posterior in the stroma of the rabbit cornea. Surface epithelial desquamation can also be studied and the effects of contact lens use can be demonstrated. The instrument is also useful for diagnosing and guiding therapy for some human diseases such as Acanthamoeba keratitis. Colonies of bacteria may also be observed and treatment evaluated in patients with infectious crystalline keratopathy. Confocal microscopy can also image the retina.

Contact Lenses↗