Rahul R. Mohan

Apoptosis, necrosis, proliferation, and myofibroblast generation in the stroma following LASIK and PRK.

The aim of this study was to semi-quantitatively analyze stromal cell apoptosis, stromal cell proliferation, and myofibroblast generation over time points from 4hr to 3 months in rabbit eyes having photorefractive keratectomy (PRK) or laser in situ keratomeliusis (LASIK). Stromal cell necrosis and inflammatory cell infiltration were also studied. PRK for low myopia (-4.5diopters [D]), PRK for high myopia (-9.0D), and LASIK for high myopia (-9.0D) were performed in rabbit eyes, and corneas were obtained for examination at 4, 24, and 72hr, 1 and 4 weeks, and 3 months after surgery. A total of 144 rabbits were included in the study. Stromal cell apoptosis, proliferation, and myofibroblast generation were evaluated semi-quantitatively by TUNEL assay, immunocytochemical analysis of Ki67, and immunocytochemical analysis of alpha-smooth muscle actin, respectively. Stromal cell necrosis and characteristics of other cell types in the stroma were evaluated by electron microscopy. Keratocyte apoptosis and the subsequent proliferation and generation of myofibroblasts were qualitatively and quantitatively different in PRK for high myopia compared to either PRK for low myopia or LASIK for high myopia. Stromal cell necrosis becomes a significant form of cell death by 24hr after injury and may involve corneal fibroblasts, myofibroblasts, and inflammatory cells. Large numbers of polymorphonuclear cells and monocytes invade the cornea by 24hr after surgery and persist for over 1 week. The qualitative and quantitative differences in the cellular wound healing response after PRK for high and low myopia and LASIK for high myopia are likely determinants of the clinical differences in refractive outcome and some of the complications, such as regression and haze, seen after these procedures.

Gene transfer into rabbit keratocytes using AAV and lipid-mediated plasmid DNA vectors with a lamellar flap for stromal access.

Development of gene transfer methods that can precisely deliver therapeutic genes to the localized or targeted tissue(s) would be highly beneficial in developing new gene therapy approaches and may also extend animal models for studying in vivo gene function and regulation at molecular levels in the selected tissues. We investigated lipid- and AAV-mediated gene transfer in rabbit cornea using a lamellar flap-technique. The goals of this study were to (1) analyze methods for in situ gene transfer into keratocytes, (2) identify efficient and suitable vectors for gene transfer into keratocytes, and (3) characterize times of first detectable expression, localization and duration of transgene expression in keratocytes with different vectors. A lamellar flap was produced in the rabbit cornea with a microkeratome. Recombinant adeno-associated viral vector (rAAV) expressing either beta-galactosidase (rAAV-beta-gal) or chloramphenicol acetyltransferase (rAAV-CAT) reporter genes, or plasmid-cationic lipid complexes expressing CAT (pMP6-CAT) or beta-galactosidase (pTR-beta-gal) were applied beneath the lamellar flap for two minutes. The flap was repositioned and eyelids sutured overnight. Corneas were removed at 4hr, 12hr, 36hr, 3 days, 7 days, or 10 days after application and either fixed in 2% formaldehyde, cryosectioned and stained for beta-galactosidase activity or homogenized and measured for CAT levels by ELISA. Corneas infected with rAAV-beta-gal vector showed positive beta-gal staining in the center and periphery of the flap interface in whole corneas and corneal beds at 3, 7, and 10 days, but not at earlier time points. Corneas treated with pTR-beta-gal plasmid vector showed positive beta-gal expression at the interface at 4, 12 and 36hr, but not at 3 or 7 days. The posterior surface of the lamellar interface where the vector was applied showed more expression than the overlying anterior surface with both plasmid and viral vectors. The level of gene expression was less with plasmid vector than viral vector monitored using beta-gal staining. CAT-ELISA confirmed expression of the CAT reporter gene with either the plasmid or rAAV vector. These results demonstrate that foreign genes can be introduced into keratocytes with plasmid or viral vectors using a lamellar flap to gain access to the stroma. The expression profile of the reporter genes depended on the vector. Transfection of keratocytes with plasmid vectors produced rapid expression of the reporter genes, but for a short duration. Reporter gene expression following transduction by rAAV vector was delayed several days, but was at higher levels and for a longer duration. This is the first report to demonstrate selective gene transfer into keratocytes and would be highly useful in studying function and regulation of genes in vivo and may eventually furnish a tool for the treatment of corneal dystrophies.

Development of genetically engineered tet HPV16-E6/E7 transduced human corneal epithelial clones having tight regulation of proliferation and normal differentiation

Lack of an optimal in vitro model of human corneal epithelial (HCE) cells is a major limitation in studying normal functions and gene regulations in HCE. Moreover, availability of a multi-layered HCE culture can reduce the usage of animals in the toxicity testing of consumer products. We have developed tetracycline-responsive human papilloma virus (HPV) 16-E6/E7 transduced HCE clones showing tight regulation of proliferation and normal differentiation. Expression of HPV16-E6/E7 mRNA and HPV16-E7 and keratin K3 proteins was examined by RNase protection assay and western blotting, respectively, in presence and absence (+/-) of Dox in identified clones. Localization of cornea-specific keratin k3 in +/- of Dox was evaluated by immunocytochemistry. The response of growth factors such as hepatocyte growth factor (HGF) and epidermal growth factor to the cellular proliferation in +/- of Dox in the newly identified clones was measured by cell counting. Cellular morphology, formation of multi-layered cultures at air-liquid interface and ultrastructural features were evaluated by light and transmission electron microscopy. The physical barrier established by the newly developed clones was determined by the transepithelial permeability to sodium fluorescein and transepithelial electrical resistance assays in the airlifted-stratified cultures.

Effect of ectopic epithelial tissue within the stroma on keratocyte apoptosis, mitosis, and myofibroblast transformation

The purpose of this study was to examine the effects of the epithelium on processes involved in stromal wound healing. Lamellar epithelial-stromal flaps were produced in rabbit corneas with a microkeratome. Peripheral corneal epithelial tissue, central corneal epithelial tissue, or no epithelial tissue (control) was introduced beneath the flap. Corneas were removed at time points from 4 hr to 1 month after surgery. Tissue sections were analyzed with immunocytochemistry for Keratin 3 (K3) to detect epithelial antigen, terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick-end labelling (TUNEL) assay to detect apoptosis, immunocytochemistry for Ki67 to detect cell proliferation, and immunocytochemistry for alpha-smooth muscle actin (SMA) to detect myofibroblasts. K3 was detected at the level of the interface from 4 hr to 1 month after surgery in corneas in which epithelial tissue was introduced, but not control corneas, with the exception of one that developed epithelial in growth. Keratocyte apoptosis was significantly higher at 4 hr after flap formation in both groups in which corneal epithelial tissue was introduced beneath the flap compared with controls. Keratocyte proliferation was significantly greater at 72 hr in corneas in which epithelial tissue was introduced beneath the flap compared to the controls. Corneas in which epithelial tissue was introduced into the interface, but not control corneas, had stromal cells expressing alpha-SMA in the stroma anterior and posterior to the interface at 1 week and 1 month after surgery. This was also noted in the control cornea in which there was epithelial ingrowth. Signals derived from the corneal epithelium promote keratocyte apoptosis. Keratocyte proliferation is higher in corneas that have lamellar surgery when epithelial tissue is introduced into the interface. Epithelium-derived signals also participate in the generation and/or maintenance of myofibroblasts in the corneal stroma.

Apoptosis in the Cornea in Response to Epithelial Injury: Significance to Wound Healing and Dry Eye

Chronic ocular surface injury is a hallmark of dry eye disease. The extent of objective surface abnormality is highly variable between patients, and roughly correlates with the severity of the disease. These surface changes extend from mild conjunctival rose bengal staining to diffuse conjunctival and corneal rose bengal and fluorescein staining. Previous studies have demonstrated that corneal surface injury triggers keratocyte apoptosis and a subsequent wound healing response in the stroma. However, the majority of patients with mild to moderate surface abnormalities from dry eye have no evidence of stromal haze, unstable refractive error, or other changes that would be expected if significant ongoing stromal wound healing were triggered by the surface changes. Superficial injury to the corneal or conjunctival epithelium probably does not induce stromal wound healing because of the intact barrier function of the epithelium and lack of penetration of proapoptotic cytokines into the corneal stroma and subconjunctival tissues. New data suggest in response to IL-1 or tumor necrosis factor (TNF) alpha is monocyte chemotactic and activating factor (MCAF)18 (also Hong, Liu, and Wilson, unpublished data, 2000). This upregulation of MCAF in response to IL-1 or TNF alpha stimulation was confirmed by RNAse protection assay (mRNA) and western blotting (protein). MCAF has chemotactic effects on monocyte and macrophage cells. This observation has led us to formulate a model to explain factors attracting inflammatory cells into corneal stroma or subconjunctival connective tissue in response to epithelial injury. This hypothesis holds that: 1) Significant injury to the epithelium (mechanical or other) results in the release of IL-1 and TNF alpha from the epithelial cells. 2) If the injury to the epithelium is sufficient to break down the epithelial barrier function, then IL-1 and TNF alpha penetrate into the stroma or subconjunctival tissues. Some types of injury might directly trigger release of IL-1 or TNF alpha from the basal epithelial cells. 3) IL-1 or TNF alpha that penetrates into the subepithelial tissues binds to receptors expressed by keratocytes or conjunctival fibroblasts, and triggers MCAF production. 4) MCAF attracts and activates macrophages. Work is in progress to characterize the effects of MCAF. We also hope to identify other cytokines released by keratocytes or conjunctival fibroblasts that attract T cells and polymorphonuclear cells in response to epithelial injury or other pathophysiological triggers.