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Purpose: The conventional Slc4a11 knockout (KO) shows significant corneal edema at eye opening, a fact that complicates the study of the initial events leading to edema. An inducible KO would provide opportunities to examine early events following loss of Slc4a11 activity., Methods: Slc4a11 Flox (SF) mice were crossed with mice expressing the estrogen receptor Cre Recombinase fusion protein and fed tamoxifen (Tm) for two weeks. Corneal thickness (CT) was measured by OCT. At eight weeks endpoint, oxidative damage, tight junction integrity, stromal lactate concentration, endothelial permeability, differentially expressed transporters, and junction proteins were determined. Separately, a keratocyte only inducible Slc4a11 KO was also examined., Results: At four weeks post-Tm induction Slc4a11 transcript levels were 2% of control. Corneal thickness increased gradually and was 50% greater than Wild Type (WT) after eight weeks with significantly altered endothelial morphology, increased nitrotyrosine staining, significantly higher stromal lactate, decreased expression of lactate transporters and Na-K ATPase activity, higher ATP, altered expression of tight and adherens junctions, and increased fluorescein permeability. No significant differences in CT were found between WT and keratocyte only Slc4a11 KO., Conclusions: The Slc4a11 inducible KO shows development of a similar phenotype as the conventional KO, thereby validating the model and providing a tool for further use in examining the sequence of cellular events by use of noninvasive in vivo physiological probes.

Purpose: We analyzed the role of N-cadherin in maintaining proper architecture and function of corneal endothelium., Methods: To achieve specific removal of N-cadherin from corneal endothelium, we bred mice carrying a floxed N-cadherin gene with those expressing the Cre-recombinase gene under the control of P0 promoter. The corneal structure was analyzed by immunostaining for cell junction proteins as well as by electron microscopy. The apoptotic status was assessed by TUNEL staining. The permeability of corneal endothelium was evaluated using fluorescein dye., Results: Removal of endothelial N-cadherin led to the appearance of opacity in the adult corneas. All corneal layers exhibited histological defects: The apical junctional complex (AJC) in corneal endothelium was disorganized, losing the continuity in tight junctions. Collagen fibrils were rearranged in the stroma. The corneal epithelium showed decreased thickness and TUNEL staining revealed increased central areas of apoptosis. Fluorescein dye injection in the anterior chamber confirmed an increased permeability of the endothelial layer. Developmental analysis indicated that, although N-cadherin was lost during embryonic stages, the AJC was maintained normally until early postnatal stages, probably due to the presence of other cadherins at these developmental stages. The junctional defects in endothelial cells, however, became obvious by postnatal day 21 (P21), although stromal and epithelial phenotypes were clearly detectable only in the adult eyes., Conclusions: N-cadherin is essential for maintaining proper structure of corneal endothelial AJCs from late postnatal to adult stages. Its ablation leads to increased endothelial permeability and corneal edema in mature eyes.

Purpose: Klf4, one of the highly expressed transcription factors in the mouse cornea, plays an important role in maturation and maintenance of the ocular surface. In this study, the structure and proteoglycan composition of the Klf4 conditional null (Klf4CN) corneal stroma was investigated, to further characterize the previously reported Klf4CN stromal edema., Methods: Collagen fibril spacing and diameter were calculated from scattering intensity profiles from small angle synchrotron x-ray scattering patterns obtained across the cornea along a vertical meridian at 0.5-mm intervals. Collagen fibril organization and proteoglycans were visualized by electron microscopy (EM), with or without the cationic dye cuprolinic blue. Proteoglycans and glycosaminoglycans were further analyzed by fluorophore-assisted carbohydrate electrophoresis (FACE) and immunoblot analysis. Q-RT-PCR was used to measure the transcript levels., Results: In the central cornea, the average collagen interfibrillar Bragg spacing increased from 44.5 nm (SD +/-1.8) in wild-type to 66.5 nm (SD +/-2.3) in Klf4CN, as measured by x-ray scattering and confirmed by EM. Mean collagen fibril diameter increased from 32 nm (SD +/-0.4) in wild-type to 42.3 nm (SD +/-4.8) in Klf4CN corneal stroma. Downregulation of proteoglycans detected by EM in the Klf4CN stroma was confirmed by FACE and immunoblot analysis. Q-RT-PCR showed that, whereas the Klf4CN corneal proteoglycan transcript levels remained unchanged, matrix metalloproteinase (MMP) transcript levels were significantly upregulated., Conclusions: The Klf4CN corneal stromal edema is characterized by increased collagen interfibrillar spacing and increased diameter of individual fibrils. The stroma also exhibits reduced interfibrillar proteoglycans throughout, which is possibly caused by increased expression of MMPs.