W. M. Petroll

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.

Meibomian gland dysfunction in chronic blepharitis

We examined 57 patients with symptoms of chronic blepharitis using meibomian gland expression, meibography, tear osmolarity, and the Schirmers test. We also performed meibography on 20 normal patients free of chronic blepharitis. We found that 42 blepharitis patients (74%) had evidence of meibomian gland loss, whereas only four of 20 normal patients (20%) had any gland dropout. We performed cluster analysis on the data from the patients with blepharitis and found that these patients tended to fall into distinct groups with clinically relevant characteristics. We also found that tear osmolarity correlated positively with gland dropout (+0.413) and negatively with excreta volume (−0.499). This study demonstrates that an objective analysis of meibomian gland function may be used to assess chronic blepharitis and define subsets of blepharitis with measurable differences. It also supports the significance of meibomian gland dysfunction on tear osmolarity and the evaporative state of the eye.

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.

Meibomian gland morphology and tear osmolarity: changes with Accutane therapy.

We evaluated the meibomian gland function of 11 patients before and during treatment with isotretinoin (Accutane) by assessing tear osmolarity, meibomian gland morphology, tear production, rose bengal staining, and meibomian gland excreta. We found, during Accutane use, that meibomian glands appeared significantly less dense and atrophic by meibography. Excreta thickness increased from 1.7 ± 0.9 to 3.1 ± 1.2 (p < 0.005), and expressible excreta volume decreased from 1.52 ± 0.68 to 1.10 ± 0.3 (p < 0.05) (scale 1-4). We also found a significant increase in tear osmolarity from 304.9 ± 11 to 316.3 ± 10 mosmol/L (p < 0.005). There was no significant change in the Schirmer test during treatment. We suggest that the clinical symptoms of blepharitis during Accutane therapy are related to decreased meibomian gland function and consequent increased tear evaporation and tear osmolarity.

Comparison of in vivo and ex vivo cellular structure in rabbit eyes detected by tandem scanning microscopy

Using the tandem scanning microscope, in vivo confocal microscopic images of living eyes were compared to images obtained from ex vivo, freshly enucleated or fixed tissue in the rabbit. In the normal cornea, microscopic details of the superficial epithelium, basal lamina, stromal fibrocyte nuclei, nerves and endothelial cell borders were easily discernible. Removal of the eye from the intact animal resulted in loss of detail with distortion of the normal structural interrelationships within the corneal stroma whilst enhancing details of the corneal epithelium. Formalin fixation further enhanced details of the basal and suprabasal corneal epithelial cell nuclei and the stromal fibrocyte cell borders whilst inducing prominent brightly reflecting folds in the thickened stroma with concomitant enhancement of the edge contrast of the collagen lamellae. These changes appeared to be related, in part, to hydration of the cornea and artefactual pooling of water between structures that may enhance reflectivity by increasing the difference between the refractive index of the cellular and extracellular elements. We conclude that microscopic examination of ex vivo preparations of corneal tissue, although providing increased resolution similar to conventional light microscopic techniques, significantly altered the normal structural relationships and could lead to erroneous measurements of the physiological properties of the tissue as compared to in vivo microscopy of undisturbed, intact tissue.

In vivo confocal microscopic studies of endothelial wound healing in rabbit cornea.

Corneal endothelial wound healing in living rabbit eyes after mechanical scrape (MS) and transcorneal freeze (TCF) injury was studied using tandem scanning confocal microscopy (TSCM). MS injury was created on the central corneal endothelium with an olive tip cannula; TCF injury was created using a 3-mm-diameter stainless steel probe cooled with liquid nitrogen. In vivo observation of wound healing using TSCM was correlated with scanning electron microscopy (SEM) for fixed tissues. At 6 h after MS, migrating endothelial cells at the leading edge showed lamellipodial processes on in vivo TSCM and SEM. After 24 h, the denuded area was almost fully resurfaced by migrating endothelial cells showing wide spaces between nuclei by TSCM. After 28 days, resurfaced endothelial cells showed normal hexagonal mosaic appearance with enlarged cells by TSCM and SEM. TCF injury produced fibroblastic changes in the endothelial cells with elongation and spreading by 24 h after injury. After 3 days, the wounded area was resurfaced with two cell types: (a) migrating endothelial cells at the peripheral area, which appeared polygonal in shape with wide intracellular spaces and (b) fibroblast-like cells at the center of the wound, which formed a retrocorneal fibrous membrane (RCFM). The RCFM was posteriorly covered with normal endothelium after 28-60 days. TSCM of the stroma demonstrated spindle-shaped, activated keratocytes migrating into the wounded stroma at 3-14 days. In conclusion, TSCM allows viewing of dynamic four-dimensional morphologic changes (x, y, z, and time) during in vivo cellular repair of corneal wound healing after either MS or TCF injury.

Quantitative assessment of anteroposterior keratocyte density in the normal rabbit cornea

The anteroposterior keratocyte density distribution in the rabbit cornea was measured. Unsectioned tissue blocks from the central cornea of five rabbits were stained with propidium iodide and imaged using a Leica laser scanning confocal microscope. A z-series of images was acquired in each sample, from anterior to posterior stroma in either 3− or 8-μm steps. Software was developed to allow interactive marking of the keratocyte nuclei within each section of the z-series and for calculating cell density. For convenience, cell density was expressed as the number of cells per corneal volume element (CVE), where CVE is a newly defined volume unit with x, y, and z dimensions of 250, 250, and 10 μm, respectively. The calculated keratocyte density was 20.2 ± 1.0 cells/CVE (n = 5), which is equivalent to 32,360 ± 1,660 cells/mm3. The greatest density was underneath the epithelium (26.3 ± 2.5 cells/CVE), the density then decreased linearly with depth to 15.2 ± 1.4 cells/CVE; there was a slight increase in density pre-Descemets membrane to 18.5 ± 3.5 cells/CVE. A 30% decrease in cell density over the entire anteroposterior stromal thickness was observed. To facilitate statistical analysis, the cell density was averaged over 5% thickness intervals from anterior to posterior cornea. A significant difference in mean cell density of these intervals was found (ANOVA, n = 20, p < 0.01). To further assess the density distribution, linear regression analysis was performed. A significant correlation was found between keratocyte density and stromal depth (R = −0.94, n = 20, p < 0.05). We conclude that in the rabbit, keratocyte cell density is maximal underlying the epithelium and progressively decreases from anterior to posterior cornea.

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.