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Purpose To provide insight into the mechanism underlying corneal deformation in keratoconus, we examined the relations among corneal curvature, thickness, and volume as well as the association of corneal scar formation with these parameters. Methods A total of 288 corneas of 174 keratoconus patients and 114 corneas of 57 control subjects were examined by anterior segment-optical coherence tomography (AS-OCT). Anterior and posterior refractive values, corneal thickness (CT), and corneal volume (CV) were determined by AS-OCT for both control and keratoconic eyes. The pattern of corneal stromal scarring was also determined from the AS-OCT images. Results The distribution of CV was similar for keratoconic and control eyes, whereas anterior and posterior refractive values as well as CT showed a wider distribution for keratoconic eyes. The progression of corneal deformation initially occurred without corneal thinning but was later associated with a decrease in CT and an eventual loss of CV. The progression of scarring from the anterior to the posterior stroma was associated with an increase in anterior refractive value and decreases in posterior refractive value, CT, and CV. Conclusions The progression of keratoconus as reflected by corneal deformation was associated with a reduction in CT and CV as well as stromal scar formation. The loss of CV occurred after the initial decline in CT, suggesting that stromal degradation occurred only at the advanced stage of keratoconus.

Purpose To develop and assess a method for quantitation of lower tear meniscus height (TMH) with the Kowa DR-1α tear interferometer. Methods Sixty-nine eyes of 49 men and 20 women (36 healthy volunteers, 33 patients with aqueous-deficient dry eye [ADDE]; mean age ± SD, 50.0 ± 14.0 years) were enrolled. TMH of each subject was measured by two observers both with DR-1α and newly developed software and with anterior-segment swept-source optical coherence tomography (SS-OCT). Intraoperator repeatability and interoperator and intersession reproducibility of measurements were assessed based on the within-subject SD (Sw), coefficient of variation (CV), and intraclass correlation coefficient (ICC). Agreement between the two devices was assessed by regression and Bland-Altman analysis. Results The CV for system repeatability of DR-1α was

PURPOSE. Hypoxia impairs the barrier function of the corneal epithelium. This function depends on tight junctions, of which zonula occludens (ZO)-1 is a major component. The authors have investigated the effects of hypoxia on ZO-1 localization and expression in the rabbit corneal epithelium in vivo. METHODS. A polymethylmethacrylate (PMMA) or rigid gas-permeable (RGP) lens was applied to one eye each of albino rabbits for 24 hours. The structure of the corneal epithelium was examined by in vivo confocal microscopy, and epithelial barrier function was evaluated by measurement of central corneal thickness. The distribution and expression of ZO-1 in the corneal epithelium were examined by immunofluorescence analysis and by immunoblot and reverse transcription-polymerase chain reaction analyses, respectively. RESULTS. Application of a PMMA lens, but not that of an RGP lens, resulted in a reduction in cell size at the surface of the corneal epithelium, compared with that in control eyes, and an increase in central corneal thickness. Immunofluorescence analysis revealed a continuous pattern of ZO-1 immunoreactivity around the perimeter of superficial corneal epithelial cells in control eyes or in eyes treated with an RGP lens. In contrast, the pattern of ZO-1 staining was discontinuous and patchy in eyes treated with a PMMA lens. Amounts of ZO-1 mRNA and protein in the corneal epithelium were reduced by application of a PMMA lens but not by that of an RGP lens. CONCLUSIONS. Hypoxia at the ocular surface induced the disruption of tight junctions between superficial cells in the rabbit corneal epithelium in vivo.

Purpose To investigate the effect of a peptide (PHSRN) corresponding to the second cell-binding site of fibronectin on the expression of ZO-1 in cultured human corneal epithelial (HCE) cells. Methods The effects of the PHSRN peptide on the expression of ZO-1, -2, and -3; claudin; and occludin were determined by reverse transcription-polymerase chain reaction (RT-PCR), immunoblot, and immunofluorescence analyses. Phosphorylation of mitogen-activated protein kinases (MAPKs) and the transcription factor c-Jun was assessed with a multiplex analysis system and immunoblot analysis. The barrier function of cultured HCE cells was evaluated by measurement of transepithelial electrical resistance. Results RT-PCR and immunoblot analyses revealed the PHSRN peptide increased the amounts of ZO-1 mRNA and protein in HCE cells in a concentration- and time-dependent manner. The PHSRN peptide had no effect on the expression of ZO-2, ZO-3, claudin, or occludin. Immunofluorescence microscopy showed that the PHSRN peptide did not affect the localization of ZO-1 at the interfaces of neighboring cells. The PHSRN peptide induced the phosphorylation of the MAPKs ERK, p38, and JNK as well as that of c-Jun. The upregulation of ZO-1 expression by the PHSRN peptide was blocked by inhibitors of signaling by ERK (PD098059), p38 (SB203580), or JNK (JNK inhibitor II). The PHSRN peptide had no effect on the transepithelial electrical resistance of cultured HCE cells. Conclusions The PHSRN peptide upregulated the expression of ZO-1 through activation of MAPK signaling pathways in HCE cells. This effect of the PHSRN sequence of fibronectin may contribute to the formation of tight junctions and play an important role in the differentiation of corneal epithelial cells.

Purpose Human corneas with or without stromal edema were examined by second harmonic generation (SHG) imaging microscopy to characterize stromal collagen organization. Methods Tissue buttons from 31 corneas with stromal edema and 8 normal corneas were fixed, and 3-mm(2) blocks were cut and stained with phalloidin, to visualize the cytoskeleton. The blocks were examined by SHG imaging with a laser confocal microscope and a mode-locked titanium:sapphire femtosecond laser. Samples were scanned to a depth of 150 microm from the surface of Bowman's layer, and SHG forward- and backscatter signals were collected. Phalloidin staining was detected by conventional laser confocal microscopy. The three-dimensional structure of the anterior segment of the cornea was reconstructed from stacked SHG images. Results Three-dimensional reconstruction of SHG signals showed adherence of interwoven collagen lamellae in the anterior stroma to Bowman's layer in both normal and edematous corneas. Abnormal SHG signals at the level of Bowman's layer were observed in edematous corneas; three-dimensional images revealed that these signals were actually localized above Bowman's layer and were indicative of subepithelial fibrosis. Phalloidin staining showed transdifferentiation of stromal cells into fibroblastic cells in edematous corneas. The incidence of subepithelial fibrosis or of fibroblastic cells increased beginning 12 months after the onset of clinical stromal edema. Conclusions SHG imaging of the anterior segment of edematous corneas revealed a normal appearance of interwoven collagen lamellae in the anterior stroma. The development of subepithelial fibrosis beginning 12 months after the onset of edema suggests that stromal edema may be a progressive disease.

Recent studies have shown that confocal imaging of second harmonic-generated (SHG) signals can detect corneal collagen organization. The purpose of this study was to assess whether SHG signals can detect differences in corneal fibrosis after excimer laser surface ablation (photorefractive keratectomy [PRK]).Rabbits received 9-D PRK in one eye followed by treatment with either mitomycin C (MMC) or vehicle. Corneal haze was measured by in vivo confocal microscopy before and 2, 4, 8, and 12 weeks after surgery. Animals were then killed and corneas were evaluated by visible and nonlinear confocal microscopy.PRK induced significant haze in vehicle-treated corneas that peaked at 2 weeks and remained elevated at 12 weeks after surgery. MMC treatment significantly (P0.05) reduced corneal haze at 2 weeks and was essentially normal by 12 weeks. Imaging of SHG signals in vehicle-treated eyes showed an anterior layer of collagen forming a honeycomb network blending into a dense mat of irregularly arranged collagen fibers that overlaid normal orthogonally arranged collagen lamellae. MMC treatment showed normal collagen organization at the surface. Fibrotic tissue was associated with a high cell density and alignment of intracellular actin filaments with collagen fiber bundles. In MMC-treated eyes, an anterior acellular zone overlaid a sparsely populated stroma containing isolated and enlarged keratocytes.Imaging of SHG signals provides a sensitive means for detection of corneal fibrosis after surface ablation and can be used to assess the effects of antifibrotic therapy on corneal healing after refractive surgery.