H.D. Cavanagh

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.

Corneal haze development after PRK is regulated by volume of stromal tissue removal

PURPOSE To determine whether excimer laser transepithelial photoablation can reduce the initial keratocyte loss seen after manual epithelial debridement. Second, to establish the relationship between initial depth of keratocyte and stromal loss and the subsequent development of corneal haze. METHODS Five rabbits received a 5-mm diameter monocular epithelial debridement by manual scraping. An additional five rabbits received a 5-mm diameter excimer laser transepithelial photoablation to a preset (intended) depth of 60 microns to ensure complete epithelial removal and to generate a superficial stromal keratectomy in all corneas. At various times during a 3-month. period, animals were evaluated by in vivo confocal microscopy through focusing (CMTF), which generates a quantitative image intensity depth profile of the cornea that provides measurements of (i) depth of keratocyte loss, (ii) epithelial and stromal thickness, and (iii) backscattered light from the anterior cornea as an objective estimate of corneal haze. RESULTS Manual epithelial debridement was associated with an initial loss of anterior stromal keratocytes to a depth of 108 +/- 14 microns that was followed by repopulation with migratory keratocytes. These cells showed increased reflectivity producing significant backscattering of light equivalent to clinical haze grade 1-2 (1,442 +/- 630 U) at 3 weeks. Furthermore, repopulation occurred without detectable inflammation and was associated with a rapid restoration of normal keratocyte morphology and reflectivity. Transepithelial photoablation induced complete epithelial debridement in all corneas in addition to a superficial stromal keratectomy of 14-44 microns. Photoablation induced 36% less initial keratocyte loss (69 +/- 19 microns) in the anterior stroma than manual debridement (p < 0.01) but was associated with intense concomitant inflammation. Photoablated corneas showed significantly more light backscattering (p < 0.01) compared with manually debrided corneas with a threefold increase at 3 weeks (4,397 +/- 1,367 U) and a sixfold increase at 3 months (1,483 +/- 1,172 compared with 234 +/- 91 U). Backscattering of light or haze increased proportionally with increasing stromal keratectomy depth (r = 0.95, p < 0.001) but was unrelated to depth of induced keratocyte death. The increased backscatter in photoablated corneas appeared related to (i) a more pronounced keratocyte repopulation response with a higher density and reflectivity of migratory fibroblasts and (ii) myofibroblast transformation after repopulation. CONCLUSIONS Excimer laser transepithelial photoablation induced significantly less keratocyte loss than manual epithelial debridement; however, photoablation was followed by a more intense inflammatory response and a greater increase in backscattering of light (haze) that was associated with increased keratocyte activation and myofibroblast transformation. Most important, the magnitude of corneal wound repair and the development and duration of corneal haze increased proportionally with increasing stromal photoablation depth (i.e., the volume of stromal tissue removal) but were unrelated to depth of initial keratocyte loss.

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

Previous studies have shown that TGFβ 1, induces activation and myofibroblast transformation of cultured rabbit corneal keratocytes. To determine whether TGFβ has a similar function in vivo, we evaluated the effect of TGFβ-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 μl of Celluvisc/PBS, containing 10, 50, or UK) μg of 1DI1, a mouse monoclonal anti-TGFβ-blocking antibody. Control wounds received either 100 μg 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 monocloncal antibody, in vivo corneal fibrosis averaged 100±26 (μm thick at day 14, whereas treatment with 10, 50, and 100 μg anti-TGFβ significantly reduced (p < 0.0005) the anterior disorganization in a dose-dependent fashion to 101 ± 32, 45 ± 11, and 56 ± 18 μm, respectively. Semiquantitative measurement of anti-fibronectin staining within the wound revealed that anti-TGFβ significantly reduced the intensity of anti-fibronectin staining in the anterior 50 μm of the corneal stroma (p < 0.003). These findings indicate that TGFβ plays an important in vivo role in keratocyte activation and myofibroblast transformation. Furthermore, the in vivo use of TGFβ-blocking antibody effects may allow modulation of corneal fibrosis after refractive surgery.

Confocal microscopic studies of living rabbit cornea treated with benzalkonium chloride.

&NA; The effects of benzalkonium chloride (BAK) on the living rabbit cornea were studied by in vivo Tandem scanning confocal microscopy (TSCM) and confirmed by conventional scanning electron microscopy (SEM). Two drops of saline or phosphate‐buffered saline (PBS) containing BAK in concentrations of 0.02, 0.01, and 0.005% was applied to rabbit eyes 15 times at 5‐min intervals. The solutions were pH 5.5‐5.9 (saline) and pH 7.5 (PBS), and osmolarity was 275‐280 (saline) and 300‐307 mOsm (PBS). Immediately after application of 0.02 and 0.01% BAK, no normal corneal superficial epithelial cells could be imaged by in vivo TSCM. No swelling of the superficial epithelial cells was observed for the control solution without BAK; however, there was a small amount of desquamation. Application of as little as 0.005% BAK caused the superficial epithelial cells to swell and desquamate. The observed desquamation of corneal superficial epithelial cells increased with higher BAK concentrations applied to the eye. One hour after final drug application, inflammatory cells appeared on the surface of the cornea treated with 0.02% BAK. These findings were correlated with SEM observations. Based on the results of this study, we believe that BAK used frequently can produce clinical corneal toxicity and that the cytotoxicity of any topical ophthalmic solutions can be evaluated by in vivo TSCM.

Time to Resolution of Contact Lens-Induced Corneal Warpage Prior to Refractive Surgery

PURPOSE To evaluate the resolution of contact lens-induced corneal warpage before keratorefractive surgery. METHODS We prospectively studied the eyes of 165 consecutive contact lens-wearing patients evaluated for keratorefractive surgery. Significant contact lens-induced corneal warpage was detected by comeal topography in 20 eyes of 11 patients. Manifested refraction, keratometry, and cornea topography were subsequently recorded during weekly or biweekly reevaluations and were compared with previous measurements for stability. Effects of age, sex, type, and duration of contact-lens wear and the recovery time period to stabilization were analyzed. RESULTS Overall, a 12% incidence of significant contact lens-induced corneal warpage was found. In patients demonstrating lens-associated warpage, the mean duration of prior contact lens wear was 21.2 years (range 10 to 30 years); lens use included daily wear soft (n=2), extended-wear soft (n=6), toric (n=4), and rigid gas-permeable contact lenses (n=8). Up to 3.0 diopter (D) refractive and 2.5D keratometric shifts accompanied by significant topography pattern differences were observed. The average recovery time for stabilization of refraction, keratometry (change within +/- 0.5D), and topography pattern was 7.8+/-6.7 weeks (range 1 to 20 weeks). Recovery rates differed between the lens types: soft extended-wear 11.6+/-8.5 weeks, soft toric lens 5.5+/-4.9 weeks, soft daily wear 2.5+/-2.1 weeks, and rigid gas-permeable 8.8+/-6.8 weeks. CONCLUSION We observed a 12% incidence of significant contact lens-induced corneal warpage in patients undergoing evaluation for keratorefractive surgery. Warpage occurred with all types of contact lens wear but resolved at different rates. To optimize the quality and predictability of keratorefractive procedures, an appropriate waiting period is necessary for contact lens-induced corneal warpage to stabilize. We suggest that resolution of corneal warpage be documented by stable serial manifested refractions, keratometry, and corneal topographic patterns before scheduling patients for keratorefractive surgery.

CHANGES IN CORNEAL ENDOTHELIAL APICAL JUNCTIONAL PROTEIN ORGANIZATION AFTER CORNEAL COLD STORAGE

PURPOSE Understanding the mechanisms regulating corneal endothelial permeability during storage and recovery is of critical importance both to improving Eye Banking practices and preventing corneal transplant failure. The goal of this study was to determine the effects of cold storage on the organization of apical junctional complex (AJC) proteins and their relationship to F-actin in corneal endothelium. METHODS Immunostaining using antibodies to the AJC proteins, ZO-1, cadherin, and alpha- and beta-catenin was performed on 16 eye bank corneas and four cat corneas after 2-8 days of storage at 4 degrees C in Optisol-GS, and compared with fresh corneas. The 3-D in situ localization of the AJC proteins was then determined by using laser confocal microscopy. AJC organization also was assessed after stored human corneas were further incubated at 37 degrees C in Optisol-GS or in serum-free media. RESULTS In normal human and cat corneas, F-actin was organized into dense peripheral bands (DPBs) along the apical cell border. The tight-junction protein, ZO-1, and the adherens junction proteins, cadherin and alpha- and beta-catenin, each formed a uniquely discontinuous hexagonal apical band with the largest gaps occurring at the Y-junctions between adjacent endothelial cells. In stored eye bank and cat corneas, cells lost their normal hexagonal F-actin staining pattern and appeared rounded and distorted, with increased cytoplasmic staining and incomplete and condensed DPBs. Similar distortions were observed in the apical bands of cadherin, catenin, and ZO-1 staining between endothelial cells. Gaps in staining at the endothelial Y-junctions were significantly enlarged; corresponding gaps also were observed with phalloidin staining. These changes were reversed after overnight incubation at 37 degrees C in either serum-free media or Optisol-GS. Quantitative analysis demonstrated a significant increase in the size of the Y-junctional gaps (p < 0.0001) after cold storage of cat corneas as compared with fresh corneas. CONCLUSION These results suggest that disruption of the F-actin cytoskeleton and AJC may explain, in part, the loss of function (corneal swelling) after prolonged cold storage.

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.

Effects of increasing Dk with rigid contact lens extended wear on rabbit corneal epithelium using confocal microscopy.

The effects of 24-h wear of various Dk-rigid gaspermeable (RGP) contact lenses on the rabbit corneal epithelium were studied by in vivo tandem scanning confocal microscopy (TSCM), and confirmed by scanning electron microscopy (SEM). Lenses used were polymethylmethacrylate (PMMA) (Dk/L=0), RGP experimental A lens (siloxanylmethacrylate-fluoromethacrylate- methylmethacrylate, 33), experimental B (siloxanylmethacrylate- fluoromethacrylate, 56), and experimental C (siloxanylstyrene-fluoromethacrylate copolymer, 64 x 10-9) (cm/s) (ml O2/ml mm Hg) with 0.15-mm thickness (Dk/L measured by polarograph including boundary layer effect). After 24-h PMMA lens wear, TSCM showed no superficial epithelial cells but only exposed, underlying wing cells. The cornea with experimental A showed partial superficial epithelial desquamation. With experimental B wear, slight superficial epithelial cell swelling and desquamation were observed on the surface of the cornea. No changes were observed for the eye with experimental C and control. The observed severity of desquamation of superficial epithelial cells was dependent on the oxygen transmissibility (Dk/L) of RGP lenses worn. All in vivo findings were confirmed by SEM observations. Based on the results of this study, we conclude that (a) although Dk/L=56 lens B shows no residual overnight corneal swelling, surface damage is still produced; (b) Dk/L=64 lens C is best for epithelium showing the same corneal images as control; and (c) TSCM is a good way to evaluate the contact lens safety and efficacy in vivo at the cellular level noninvasively.

Organization of junctional proteins in proliferating cat corneal endothelium during wound healing

Purpose. To evaluate for the first time cell junctional protein organization in proliferating corneal endothelial cells during in vivo wound healing. Methods. A total of 16 cats (32 eyes) were used in this study. A single 3-mm diameter (n = 24) or 1-to 2-mm diameter (n = 8) scrape injury was created in the central corneal endothelium of each eye. Twenty-four, 48, 72 hours or 5 days after scrape injury, eyes were collected for in situ double-or triple-labeling with phalloidin, anti-ZO-1, &agr;-catenin, &bgr;-catenin, and MIB-1 (monoclonal antibody to Ki67, a marker for actively cycling cells) and were imaged using confocal laser microscopy. Results. In 3-mm diameter injuries, endothelial cells completely resurfaced the wound 48 to 72 hours after scrape injury; smaller wounds resurfaced by 48 hours. Ki67 staining was negative 24 hours after scrape injury in all cases. Ki67-positive cells were observed in the central region of the wounds after 48 and 72 hours, and mitotic figures and pairs of postmitotic cells were observed. On day 5, Ki67-positive cells were rarely detected, and no mitotic figures were observed. In the wound area, a significant increase in cell area and a reduction in hexagonality were observed in cycling cells after 48 and 72 hours. Normal apical, pericellular staining of f-actin, ZO-1, &agr;-catenin, and &bgr;-catenin was partially maintained at all times during wound healing of small and large wounds. Double-labeling confirmed that these proteins were also present along the apical cell border in Ki67-positive cells. Conclusions. After in vivo scrape injury, proliferation is limited temporally and spatially to spreading endothelial cells within the wound. Cell junctional connections appear to be maintained in actively cycling cells during healing.

Effect of contact-lens-induced hypoxia on lactate dehydrogenase activity and isozyme in rabbit cornea.

Lactate dehydrogenase (LDH) levels and subunit isozyme patterns in cornea were monitored in 36 albino rabbits wearing thick, rigid, gas-permeable contact lenses for periods of 24 h, 2 and 7 days, and 1 and 3 months. The oxygen transmissibility of the contact lens was 15.3 x 10-9 (cm/s) (ml O2/ml mm Hg). The activity of LDH in corneal tissue decreased according to the duration of lens wear. The LDH isozyme patterns shifted with lens wear from LDH 1,2,3 (heart type, aerobic) to LDH4,5 (skeletal muscle type, anaerobic). The cornea swelled 8.8% with overnight contact lens wear, with increased swelling (11- 12%) after further continuous lens wear. After contact lens removal, however, the LDH activity and the isozyme pattern returned to normal, and the corneal thickness quickly returned to normal. Based on these observations, it is suggested that LDH in rabbit corneas was physiologically affected by lens-induced hypoxia, but these changes were reversible. These results might further suggest that tear LDH levels in the human contact lens wearer could provide an ongoing assessment of the tolerance of the lens by the ocular surface.