Corneal Homeostasis Research Articles

4α‐PDD induces Ca2+ influx in human corneal epithelial cells by activating TRPV4 channels

Abstract Purpose: Transient receptor potential (TRP) isoform expression is evident in human corneal epithelial cells (HCEC‐SV40). However, their role in maintaining corneal epithelial homeostasis is not fully understood. We probed for gene and protein expression as well as functional activity of the vanilloid subtype, TRPV4, in immortalized HCEC‐SV40 since they elicit Ca2+ dependent regulatory volume decrease (RVD) responses during exposure to a hypotonic challenge. Methods: RT‐PCR and Western blotting analyses identified TRPV4 gene and protein expression. Functional activity was assessed based on determining whether the TRPV4 selective agonist, 4α‐PDD, induced transients increases in intracellular Ca2+ concentration. Results: Single cell fluorescence imaging results showed that 4α‐PDD (3 μM) increased intracellular Ca2+ concentration. The fura2 fluorescence ratio (f340/f380) was 0.39 ± 0.03578 in the resting state (n = 5). After application of 4α‐PDD it increased to 0.904 ± 0.14363 (n = 5; p = 5.72077×10‐5). This increase was abolished by the TRP channel blocker ruthenium red or by Ca2+‐free Ringer's medium. Conclusions: In conclusion, there is functional TRPV4 expression in HCEC‐SV40. TRPV4 expression may provide an osmosensor role to induce RVD behavior during exposure to a hypotonic challenge since this response is mediated through intracellular Ca2+ transients.Supported in part DFG Pl 150/14‐1 and NIH, EY04795. CR: none

Existence of small slow-cycling Langerhans cells in the limbal basal epithelium that express ABCG2

Despite the obvious importance of limbal stem cells in corneal homeostasis and tumorigenesis, little is known about their specific biological characteristics. The purpose of this study was to characterize limbal slow-cycling cells based on the expression of ABCG2 and major histocompatibility complex (MHC) class II and the cell size. Wistar rats were daily injected with 5-bromo-2-deoxyuridine (BrdU) at a dose of 5 mg/100 g for 2 weeks. After 4-week BrdU-free period, corneal tissues were excised, and immunofluorescence staining for ABCG2, BrdU, and MHC class II was performed by confocal microscopy. In another series, corneal tissues of normal rat were double immunostained for ABCG2, keratin 14, keratin 3, CD11c, and MHC class II. In addition, limbal, peripheral and central corneal epithelial sheets were isolated by Dispase II digestion and dissociated into single cell by trypsin digestion and cytospin preparations were double immunostained for ABCG2 and MHC class II. The cell size and nucleus-to-cytoplasm (N/C) ratio of limbal ABCG2 + cells were analyzed and compared with those of cells from other zones. BrdU label-retaining cells (LRCs) with expression of ABCG2 were found in the limbal epithelial basal layer, but not in other parts of the cornea. Approximately 20% of these cells were MHC class II positive. All MHC class II + cells in the corneal epithelium were positive for CD11c, a marker for dendritic cells (DCs). Double labeling with ABCG2 and keratin 14 showed that nearly four-fifth of limbal ABCG2 + cells were positive for keratin 14 but negative for keratin 3, exhibiting an undifferentiated epithelial cell lineage. Cytospin sample analysis revealed the presence of a distinct population of smaller ABCG2 + cells with expression of MHC class II with a larger N/C ratio in the limbal epithelium. A new population of small slow-cycling cells with large N/C ratio has been found to express ABCG2 in the limbal epithelial basal layer. Some of these cells normally express MHC class II antigen. These findings may have important implications for our understanding of the characteristics of limbal slow-cycling cells.

Therapeutic Use of Limbal Stem Cells

Stem cells are defined as relatively undifferentiated cells that have the capacity to generate more differentiated daughter cells. Limbal stem cells are responsible for epithelial tissue repair and regeneration throughout the life. Limbal stem cells have been localized to the Palisades of Vogt in the limbal region. Limbal stem cells have a higher proliferative potential compared to the cells of peripheral and central cornea. Limbal stem cells have the capacity to maintain normal corneal homeostasis. However, in some pathological states, such as chemical and thermal burns, Stevens-Johnson syndrome, and ocular pemphigoid limbal stem cells fail to maintain the corneal epithelial integrity. In such situations, limbal stem cell transplantation has been required as a therapeutic option. In unilateral disorders, the usual source of stem cells is the contralateral eyes, but if the disease is bilateral stem cell allografts have to be dissected from family members or cadaver eyes. The advent of ex vivo expansion of limbal stem cells from a small biopsy specimen has reduced the risk of limbal deficiency in the donor eye. Concomitant immunosuppressive therapy promotes donor-derived epithelial cell viability, but some evidences suggest that donor-derived epithelial stem cell viability is not sustained indefinitely. Thus, long-term follow-up studies are required to ascertain whether donor limbal stem cell survival or promotion of recolonization by resident recipient stem cells occurs in restored recipient epithelium. However, this is not an easy task since a definitive limbal stem cell marker has not been identified yet. This review will discuss the therapeutic usage of limbal stem cells in the corneal epithelial disorders.

Muscarinic acetylcholine receptor subtypes in human corneal epithelium and endothelium

Muscarinic acetylcholine receptors are located throughout the body. The demonstration of muscarinic receptors in corneal tissue has been inconsistent. Using freshly fixed human corneal tissue, we show a complete profile of muscarinic receptor subtypes in human corneal epithelium and endothelium. Muscarinic receptor sites were studied using immunocytochemistry, immunofluorescence and immunoblotting. Antibodies to M2, M4 and M5 muscarinic receptor subtypes bound in human corneal epithelium and endothelium. No binding was found for antibodies to M1 and M3 muscarinic receptor subtypes. Our studies indicate the presence of M2, M4 and M5 muscarinic acetylcholine receptor subtypes in human corneal epithelium and endothelium. These receptors may play a role in the regulation of corneal homeostasis, other functions, like wound healing, or the pathogenesis of corneal diseases.

Silicone Hydrogel Contact Lenses and the Ocular Surface

For 30 years, contact lens research focused on the need for highly oxygen-permeable (Dk) soft lens materials. High Dk silicone hydrogel contact lenses, made available in 1999, met this need. The purpose of this review is to examine how silicone hydrogel lens wear affects the ocular surfaces and to highlight areas in which further research is needed to improve biocompatibility. Silicone hydrogel lenses have eliminated lens-induced hypoxia for the majority of wearers and have a less pronounced effect on corneal homeostasis compared to other lens types; however, mechanical interaction with ocular tissue and the effects on tear film structure and physiology are similar to that found with soft lens wear in general. Although the ocular health benefits of silicone hydrogel lenses have increased the length of time lenses can be worn overnight, the risk of infection is similar to that found with other soft lens types, and overnight wear remains a higher risk factor for infection than daily wear, regardless of lens material. Future contact lens research will focus on gaining a better understanding of the way in which contact lenses interact with the corneal surface, upper eyelid, and the tear film, and the lens-related factors contributing to infection and inflammatory responses.

Bcl-2 and Bax Regulation of Corneal Homeostasis in Genetically Altered Mice

The antiapoptotic protein Bcl-2 and its proapoptotic counterpart Bax may play an important role in the regulation of corneal epithelial renewal. To understand the physiologic importance of Bcl-2 and Bax in the healthy cornea, epithelial and stromal thickness, proliferation, and surface cell exfoliation rates were examined in the central cornea of genetically altered mice overexpressing Bcl-2 and lacking Bax. Adult Bcl-2 transgenic (n = 55) and Bax knockout (n = 82) mice and wild-type controls were clinically prescreened at no less than 2 months of age for any ocular developmental abnormalities. Polymerase chain reaction was used to confirm genotype. Corneal epithelial renewal was examined by in vivo tandem scanning confocal microscopy to measure total corneal and sublayer thickness and by immunohistochemistry on whole mount corneal tissues to determine basal epithelial cell proliferation using 5-bromo-2-deoxyuridine (BrdU) and surface cell exfoliation with a Calcein AM-Ethidium homodimer assay (live/dead). Stained corneas were scanned with a laser scanning confocal microscope, and images were digitized, and cell counts were obtained. Levels of Bcl-2 protein were assessed by Western blots. No significant changes in proliferation or cell death were found in either group compared to wild-type littermate controls; however, epithelial and stromal thicknesses were greater in the Bcl-2 transgenic group compared to wild-type and decreased in the Bax knockout. Western blotting confirmed that there was no change in the level of Bcl-2 expression in the corneal epithelium in the Bcl-2 transgenic or Bax knockout strains. The significant difference in epithelial and stromal thickness suggests a functional role for Bcl-2 and Bax in the maintenance of corneal homeostasis; however, the lack of significant alterations in proliferation, cell exfoliation, and levels of Bcl-2 protein in the adult corneal epithelium suggest that these phenotypic changes are a result of a new stable homeostatic equilibrium (proliferation + migration = shedding). Additional experiments to delineate the role of Bcl-2 and Bax during development and in the adult cornea are necessary.

Monoamine receptors in human corneal epithelium and endothelium

Monoamine receptors are found throughout the body. Reports about the presence of monoamine receptors in the human cornea are inconsistent. Immunohistochemistry, immunofluorescence and immunoblotting were used to localize monoamine receptor sites on human corneal epithelium and endothelium. Antibodies to alpha-1, beta-1 and beta-2 adrenergic receptors and to D1-like and 5HT-7 receptors were bound in corneal epithelium. Antibodies to alpha-1, alpha-2A, beta-1 and beta-2 adrenergic receptors and to 5HT-7 receptors were bound in corneal endothelium. Our data demonstrate the presence of several monoamine receptors in the human cornea. These receptors may play a role in the regulation of fluid transport or corneal homeostasis.

A Dataset of Human Cornea Proteins Identified by Peptide Mass Fingerprinting and Tandem Mass Spectrometry

Diseases of the cornea are extremely common and cause severe visual impairment worldwide. To explore the basic molecular mechanisms involved in corneal health and disease, the present study characterizes the proteome of the normal human cornea. All proteins were extracted from the central 7-mm region of 12 normal human donor corneas containing all layers: epithelium, Bowman's layer, stroma, Descemet's membrane, and endothelium. Proteins were fractionated and identified using two different procedures: (i) two-dimensional gel electrophoresis and protein identification by MALDI-MS and (ii) strong cation exchange or one-dimensional SDS gel electrophoresis followed by LC-MS/MS. All together, 141 distinct proteins were identified of which 99 had not previously been identified in any mammalian corneas by direct protein identification methods. The characterized proteins are involved in many processes including antiangiogenesis, antimicrobial defense, protection from and transport of heme and iron, tissue protection against UV radiation and oxidative stress, cell metabolism, and maintenance of intracellular and extracellular structures and stability. This proteome study of the healthy human cornea provides a basis for further analysis of corneal diseases and the design of bioengineered corneas.

Nanoscale topography modulates corneal epithelial cell migration

The purpose of this study was to evaluate the effect of surface topographic features that mimic the corneal epithelial basement membrane on cell migration. We used electron-beam and X-ray lithography and reactive ion etching to pattern silicon wafers with pitches (groove width plus ridge width) of nano- and microscale dimensions (pitches ranged from 400 to 4000 nm). Additionally, polyurethane patterned surfaces were created by replication molding techniques to allow for real-time imaging of migrating cells. Individual SV40-transformed human corneal epithelial cells frequently aligned with respect to the underlying surface patterns and migrated almost exclusively along grooves and ridges of all pitches. Direction of migration of individual cells on smooth surfaces was random. In cell dispersion assays, colonies of cells migrated out from initially circular zones predominantly along grooves and ridges, although there was some migration perpendicular to the ridges. On smooth surfaces, cells migrated radially, equally in all directions, maintaining circular colony shapes. We conclude that substratum features resembling the native basement membrane modulate corneal epithelial cell migration. These findings have relevance to the maintenance of corneal homeostasis and wound healing, as well as to the evolution of strategies in tissue engineering, corneal prosthesis development, and cell culture material fabrication.

Inhibition of Gap Junction–Mediated Intercellular Communication by TNF-α in Cultured Human Corneal Fibroblasts

Keratocytes are connected to each other by gap junctions, which mediate intercellular communication and contribute to maintenance of corneal homeostasis. The possible effect of tumor necrosis factor (TNF)-alpha, a proinflammatory cytokine, on gap junctional intercellular communication (GJIC) in cultured human corneal fibroblasts was examined. GJIC activity was measured by observing the intercellular diffusion of the fluorescent dye Lucifer yellow. The expression of the gap junction protein connexin43 (Cx43) was evaluated by immunofluorescence and immunoblot analyses with a specific monoclonal antibody. The abundance of Cx43 mRNA was determined by quantitative reverse transcription and polymerase chain reaction analysis. TNF-alpha induced a time- and concentration-dependent decrease in GJIC activity in human corneal fibroblasts. Immunofluorescence analysis revealed that TNF-alpha reduced the level of specific staining for Cx43 at sites of contact between adjacent cells. Immunoblot analysis detected four specific Cx43 bands, one corresponding to the nonphosphorylated form of the protein and three corresponding to phosphorylated forms. Exposure of cells to TNF-alpha reduced the relative abundance of the three phosphorylated forms of Cx43. The amount of Cx43 mRNA was not affected by TNF-alpha. TNF-alpha inhibited GJIC in cultured human corneal fibroblasts, an effect that was possibly mediated by dephosphorylation and consequent degradation of Cx43. The downregulation of GJIC among keratocytes in response to TNF-alpha may contribute to the breakdown of corneal homeostasis during corneal inflammation.

The corneal stroma during contact lens wear

Recent technological advances have lead to novel descriptions of the microanatomy of the corneal stroma. In the first section of this review, these findings and the role they play in the maintenance of vital properties such as corneal transparency, mechanical strength, homeostasis, wound-healing response and metabolism are described. In the second part, contact lens induced stromal alterations such as acidosis, oedema, striae, thinning and opacities are reviewed as well as the more recently described phenomenon of microdot deposits and keratocyte loss with an emphasis on how lens wearing stromal effects can be minimised.

Non-apoptotic desquamation of cells from corneal epithelium: putative role for Muc4/sialomucin complex in cell release and survival.

Muc4/sialomucin complex (SMC), a large heterodimeric mucin composed of an extracellular mucin subunit ASGP-1 and a transmembrane subunit ASGP-2, is present at the rat ocular surface localized mainly to the most superficial layers of the epithelia. To investigate corneal homeostasis and the functions of Muc4/SMC at the ocular surface, we developed a corneal epithelial cell culture system from corneal explants, from which migrating cells formed an epithelial sheet resembling the native epithelium with regard to microanatomy, expression of characteristic markers, cell migration, and Muc4/SMC expression. Cells migrating from the explants expressed smooth muscle actin. Proliferation was detected only on the edge of epithelial sheet in the immature epithelium and throughout the sheet in confluent cultures. Microscopy revealed that the epithelial sheet was formed from four to six layers of cells expressing keratin 3 and Muc4/SMC in forms identical to those expressed at ocular surface in vivo. Electron microscopy showed cells in various morphological states in the process of releasing from the surface of the multilayer (desquamating). Surprisingly, few of these cells showed evidence of apoptosis, either by morphological or DNA fragmentation analyses. These results suggest a new model for desquamation from stratified epithelia, in which desquamation and apoptosis are independent and sequential processes. Desquamating cells also exhibit a high level of Muc4/SMC. Since Muc4/SMC has been shown to be a potent anti-adhesive and a repressor of apoptosis, we propose that it plays a role in the non-apoptotic desquamation process.

Transduction of functionally active TAT fusion proteins into cornea

A technology has recently been developed that allows for the rapid transduction of full-length functionally active proteins into intact tissue through intravenous injection and into cultured cells. This technology involves the fusion of an 11 amino acid sequence of the HIV TAT protein to the protein of interest. In the current investigation, we determined whether functionally active TAT fusion proteins could be transduced into intact corneas by topical application. TAT-β-galactosidase was purified from bacterial cells and applied in serial dilutions (12·5–250 n m) to cultured epithelial cells for 5 or 15 min. In addition, enucleated globes and excised corneas with or without a central 3-mm epithelial debridement were incubated with TAT-β-galactosidase for 1 or 2 hr. Excised corneas were allowed to heal in organ culture. Transduction of active β-galactosidase was detected by incubating the cells or corneas with X-gal. TAT-β-galactosidase was transduced into nearly all cultured cells in a concentration-dependent manner. When TAT-β-galactosidase was topically applied to intact corneas, only the most superficial layer of epithelium was highly transduced. When the superficial layer was removed with nitrocellulose, two to four layers of cells were transduced. In corneas with a central debridement, epithelial cells at the edge of the debridement were transduced as well as the stromal cells subjacent to the debridement. Active β-galactosidase was maintained at least 1 day in organ culture. No X-gal reaction was seen in either cells or corneas not incubated with TAT-β-galactosidase. Functionally active proteins can be efficiently transduced into corneal epithelial and stromal cells using TAT fusion protein technology. The intact epithelium provides a barrier to penetration of TAT proteins. This barrier can be overcome by disrupting the epithelium. TAT-mediated protein transduction may be extremely useful in studies of corneal wound healing and homeostasis.

The effects of low- and hyper-Dk contact lenses on corneal epithelial homeostasis.

Contact lens were and eyelid suturing diminish overall Bcl-2 expression in the corneal epithelium, yet nuclear Bcl-2 in the exposed surface epithelial cells seems to be retained for longer periods when compared with the retention in control eyes. This observation may explain why there is a decrease in surface cell exfoliation during CTL wear, because nuclear Bcl-2 loss needs to precede apoptotic surface cell exfoliation. In addition, if the hypothesis is true that Bcl-2 also has a regulatory function in the proliferation and differentiation of tissues, the overall reduced expression of Bcl-2 (nuclear plus cytoplasmic) may clarify the decrease in proliferation of the corneal epithelium seen during CTL wear. Future studies should clarify the role of Bcl-2 in the homeostatic dynamics of the corneal epithelium. CTL wear and eyelid suturing provide excellent noninflammatory models to advance knowledge of Bcl-2 and its regulatory roles. Additionally, these models should help researchers gain a better understanding of the CTL-associated effects on corneal epithelium.

Staurosporine-induced apoptosis in human cornea epithelial cells in vitro.

Apoptosis is a an important process in corneal development, homeostasis, and disease. This study was performed to determine for the first time basic temporal apoptotic features of SV-40 immortalized human corneal epithelial (HCE) cells. Additionally, we introduce a sensitive analysis of confocal microscopic images to measure the kinetics of staurosporine (STS) induced phosphatidylserine (PS) membrane translocation and early nuclear morphological changes. HCE cells were cultured in the presence of STS to induce apoptosis. Caspase-3 activity was measured with the fluorogenic substrate z-DEVD-rhodamine 110. We determined mitochondrial viability with a 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzenedisulfonate reduction assay, and chromatin degradation with a fluorometric method using 4,6-diamidino-2-phenylindole (DAPI). Membrane translocation of PS and nuclear alterations were assessed by quantitative fluorescence microscopy. Image processing routines were written in interactive data language (IDL). Nuclear alterations like hyperchromicity, pyknosis, and active chromatin reorganization evolved instantly after STS induction. They were followed by PS translocation, DNA fragmentation, mitochondrial breakdown, and caspase-3 activation, which were detected between approximately 90 min and 4 h. Morphological and texture sensitive descriptors proved to be highly susceptible for the quantification of early apoptotic nuclear characteristics in HCE cells. We propose this method to be considered for the detection of subtle nuclear reorganization in cellular studies.

Role of oxygen in corneal epithelial homeostasis during extended contact lens wear.

Infectious corneal ulcers are seldom observed in healthy, normal eyes. Even exposing normal animal corneas to highly concentrated bacterial suspensions will not result in corneal infection.' Thus, there is evidence that the ocular surface defense system efficiently protects the eye. However, having a contact lens on the ocular surface for prolonged time impairs and alters this defense system. As a consequence, the risk of corneal infection increases, depending partially on contact lens wearing schedule, type, and oxygen transmissibility. Although the development of corneal infection is a multifactorial process, one crucial step in the pathogenesis is the penetration of microorganisms through the tightly compacted multicellular layer structure of the corneal epithelium. Based on that knowledge, we have hypothesized that maintaining a healthy functioning corneal epithelium during contact lens wear is an important and integral part in preventing and reducing the risk for infection. This review summarizes our current understanding of the corneal epithelium and its homeostasis in normal eyes during extended contact lens wear and hypoxia.

Opioid growth factor modulation of corneal epithelium: Uppers and downers

Purpose. This paper presents a concise review of the role of the intrinsic opioid growth regulation system (IOGRS) in homeostasis and wound repair of the corneal epithelium. Methods. The article is a summary of published research on the topic. Results. The native opioid peptide, [Met 5 ]-enkephalin, also termed opioid growth factor (OGF), has been demonstrated to be present in a wide variety of classes of the phylum. Chordata, and acts as an autocrine/paracrine produced, tonically active, receptor specific, negative growth modulating factor in homeostatic (uninjured) corneal epithelium in humans and non-primates. Similarly, OGF acts to down-regulate epithelial cell division and migration of corneal epithelium in the closing of corneal epithelial abrasions. Such repair can be up-regulated (hastened) in non-diabetic animals by treatment with exogenous administration of the potent opioid antagonist, naltrexone (NTX). The system also is functional in diabetic animals and can be manipulated to restore epithelial wound healing rates to normal. Conclusions. The IOGRS plays a vital role in supporting corneal epithelial homeostasis, and in modulating closure of epithelial wounds. The system should provide opportunities for novel therapies especially for corneal epithelial wound healing disorders.

Corneal epithelial homeostasis following daily and overnight contact lens wear

This report reviews and integrates our recent clinical and laboratory findings of the effects of daily (DW)/overnight (EW) contact lens wear on corneal epithelial homeostasis. In a prospective, double-masked human clinical study, three lens types (high and hyper Dk/t soft; hyper Dk/t RGP) were used to assess the effects of daily and overnight lens wear on corneal epithelial thickness, cell surface size, Pseudomonas aeruginosa (PA) binding to exfoliated epithelial cells and exfoliation rates. Concomitantly, we studied in a rabbit model, the effects of overnight lens wear on corneal epithelial surface cell viability (live/dead assay, TUNEL-labeling, Annexin-V staining, Bcl-2 expression), basal cell proliferation and migration patterns. The comprehensive results obtained yield important new insights on both the normal homeostasis of the corneal epithelium and the biological changes induced by contact lens wear.

The Epidermal Growth Factor Receptor (EGFR): Role in Corneal Wound Healing and Homeostasis

To evaluate the role of the epidermal growth factor receptor (EGFR) in corneal epithelial wound healing, the effect of an EGFR inhibitor on epithelial cell proliferation and cell stratification during wound healing was investigated. From 3 days prior to wounding until wound healing was complete, rats were systemically treated with either an EGFR tyrosine kinase inhibitor (ZD1839) at 40mgkg−1day−1or 80mgkg−1day−1, or with vehicle only (control). A single corneal wound was made in the center of 66 rat corneas, using a 6.0mm glass tube wrapped in tissue paper soaked in n-heptanol. Subsequently, each wound was photographed and measured by a computer-assisted digitizer every 12hr. To determine the number of cells in S phase, entire corneas were labelled with3H-thymidine and subjected to autoradiography at 0, 12, 24 and 48hr after wounding. Epithelial thickness was also measured at these time points by microscopy. Epithelial wound healing was significantly and dose-dependently delayed following administration of ZD1839. At 24hr after wounding, the number of S-phase cells in the limbal corneal epithelium was significantly lower in both the treated groups compared with the control group (P<0.05). In the cornea before wounding (0hr) and at 48hr post-wounding, epithelial thickness was also significantly less in treated rats compared with controls (P<0.05). These results indicate that EGFR inhibition affects epithelial cell proliferation and stratification during corneal epithelial wound healing and may play a role in maintaining normal corneal epithelial thickness.

Effect of norepinephrine on proliferation, migration, and adhesion of SV-40 transformed human corneal epithelial cells.

To determine the ability of norepinephrine to modulate proliferation, adhesion, and migration of SV-40 transformed human corneal epithelial cells. Assays were performed using SV-40 transformed human corneal epithelial cells. For proliferation assays, cells were plated in 96-well plates coated with fibronectin and collagen (FNC). A dose-response curve was generated for norepinephrine in concentrations of 100 nM-100 microM. The cell number in each well was evaluated using the fluorochrome Calcein AM (an intracellular esterase cleavage substrate), and fluorescence was determined using an automated fluorescent plate reader. For cell adhesion, 25 x 10(-3) cells were plated onto FNC-coated 96-well plates, incubated in 10 nM-100 microM norepinephrine for 90 min, gently irrigated, and the remaining adherent cells quantitated. Cell migration was measured using blind-well migration chambers with a 10-microm pore size and FNC-coated filters. Cells (250 x 10(3)) were added to the upper chamber, incubated for 18 h in the presence of factors, after which time the cells that had migrated through the filter were quantitated. The toxicity of norepinephrine was evaluated using a standard Live/Dead assay employing the combined fluorochromes of ethidium homodimer (to indicate dead cells) and Calcein AM (to indicate viable cells). Varying concentrations of norepinephrine were added, and the cells incubated for 3 h and the fluorometric assay performed. Norepinephrine stimulated corneal epithelial cell proliferation and migration over a wide range of concentrations. It did not modulate cell adhesion and demonstrated cell toxicity only at the highest (supraphysiologic) concentration tested. Norepinephrine is normally found in the cornea and may be important in the maintenance of normal corneal homeostasis and in wound-healing processes.