Jong-Soo Lee

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.

Corneal epithelial cellular dysfunction from benzalkonium chloride (BAC) in vitro.

Purpose: To investigate the functional and morphological toxicity of benzalkonium chloride (BAC) on corneal epithelial cells in vitro.

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.

Differential expression analysis by gene array of cell cycle modulators in human corneal epithelial cells stimulated with epidermal growth factor (EGF), hepatocyte growth factor (HGF), or keratinocyte growth factor (KGF)

Purpose. To identify and differentiate cell cycle and differentiation genes that are up-regulated or down-regulated in human corneal epithelial cells in response to alternative epithelium-modulating cytokines epidermal growth factor (EGF), hepatocyte growth factor (HGF) or keratinocyte growth factor (KGF). Methods. Primary cultures human corneal epithelial cell (HCE) were treated with 25 ng/ml of EGF, 25 ng/ml HGF, 25 ng/ml KGF, or vehicle for 8 hours. Complementary DNA (cDNA) probes were synthesized from total cellular RNA isolated from the HCE cells. The cDNA probes were hybridized to the Atlas human cell cycle/differentiation array membrane. RNAse protection assay was used to confirm up-regulation of the serine/threonine-protein kinase PITALRE gene by EGF, KGF, and HGF. Results. The expression of one hundred and eleven cell cycle and differentiation genes was monitored with the gene array system. It was found that these epithelial cell-modulating cytokines shared similar effects on some of the cell cycle and differentiation genes that were monitored, but had specific effects on some cytokines. Up-regulation of PITALRE gene expression was confirmed using RNAse protection assay. Conclusion. EGF, HGF and KGF had differential effects on cell cycle- and differentiation-related gene expression in corneal epithelial cells. For example, all three mitogenic growth factors up-regulated the expression of cyclin D1 (BCL-1 oncogene) and serine/threonine-protein kinase PITALRE in the primary cultured human corneal epithelial cells. However, EGF and KGF, but not HGF, up-regulated expression of the E2F-1 pRB-binding protein gene. Thus, while these three epithelial mitogens have similar effects on many genes that were analyzed, important differences were noted that may relate to differing effects of these growth factors on corneal epithelial cells. Studies to analyze the significance of the identified differences among these growth factors are in progress.

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.

Intraocular pressure and influencing systemic health parameters in a Korean population

Aim: To evaluate the relationship between intraocular pressure (IOP) and systemic health parameters such as age, gender, body mass index (BMI), total cholesterol, high density lipoprotein (HDL), and triglyceride (TG) in a Korean population. Materials and Methods: A total of 30,893 healthy subjects underwent automated multiphasic tests, including non-contact tonometry, automated perimetry, fundus photography, and blood samplings for total cholesterol, HDL, and TG. Seven age groups were divided by decades ranging from 20 to 29 years to 80 + years. The association between IOP and BMI, plasma lipid profiles was examined using cross-sectional analysis. Results: The mean age of subjects was 47.7 years. The mean IOP of subjects was 15.4 ± 3.2 mmHg for both eyes. The mean IOP of men was significantly higher than women (P = 0.000). By multiple linear regression analysis, IOP was positively associated with gender (male), BMI, total cholesterol, and TG and negatively associated with age (P = 0.000). BMI, total cholesterol, and TG had significantly positive correlations with IOP after adjusting for age, gender, and other variables which can influence the IOP (P = 0.000). Conclusions: In a Korean population, the mean IOP, total cholesterol, TG, and BMI values of men were higher than women. IOP was found to increase with total cholesterol, TG, BMI, and to decrease with only age regardless of sex.

Corneal Endothelial Changes as a Clinical Diagnostic Indicator of Dentatorubropallidoluysian Atrophy

Objective To present a rare case of patient diagnosed with dentatorubropallidoluysian atrophy (DRPLA) accompanied by corneal endothelial cell loss. Methods A 37-year-old man with choreoathetoid movement and cerebellar ataxia was diagnosed with DRPLA based on a DNA analysis compared with that of healthy control subjects. We examined the best corrected visual acuity, color vision, light reflex, topography, corneal thickness, fundus, fluorescein angiograpic findings, the visual field, ERG, specular microscopy as well as MRI and serologic tests. Results The best corrected visual acuity was 20/20 in both eyes by Snellen chart, and the other ocular findings were within normal limits except for a significantly decreased corneal endothelial cell density, 876 cells/mm2 in the right eye and 941 cells/mm2 in the left eye. Conclusions A patient with neurodegenerative disorders such as choreathetoid movement, myoclonic seizure, cerebellar ataxia, and dementia should be examined specifically by specular microscopy because corneal endothelial cell loss is the only clinical diagnostic indicator of DRPLA.

Subperiosteal hematoma after surgical treatment for subarachnoid hemorrhage.

After the surgical treatment for subarachnoid hemorrhage, computed tomography and magnetic resonance imaging clearly demonstrated a fracture in the medial wall of the left orbit and a superonasal subperiosteal hematoma. Removal of the subperiosteal hematoma and correction of the fracture were successful. Subperiosteal hematoma is an unusual complication after inadvertent neurosurgical manipulation.