Functional human corneal equivalents constructed from cell lines.

Human corneal equivalents comprising the three main layers of the cornea (epithelium, stroma, and endothelium) were constructed. Each cellular layer was fabricated from immortalized human corneal cells that were screened for use on the basis of morphological, biochemical, and electrophysiological similarity to their natural counterparts. The resulting corneal equivalents mimicked human corneas in key physical and physiological functions, including morphology, biochemical marker expression, transparency, ion and fluid transport, and gene expression. Morphological and functional equivalents to human corneas that can be produced in vitro have immediate applications in toxicity and drug efficacy testing, and form the basis for future development of implantable tissues.

Loss of keratocyte ion channels during wound healing in the rabbit cornea.

PURPOSE: Corneal keratocytes are responsible for repairing the corneal stromal matrix after injury or infection. Recent work has characterized the primary voltage-gated ion currents in keratocytes from normal, uninjured corneas. The purpose of the present study was to examine and characterize keratocyte voltage-gated ion currents from freeze-wounded rabbit corneas. METHODS: Rabbit corneas were injured using a liquid nitrogen cooled brass probe. Keratocytes were isolated from control eyes, trephined buttons of stroma encompassing the wound area, and the stromal rim surrounding the button. Ionic currents were examined using the amphotericin perforated-patch variation of the whole cell patch clamp technique. RESULTS: The delayed rectifier K+ current, described previously as the primary voltage-gated outward current in keratocytes, was found in 100% of control cells, 91% of cells isolated from the corneal rim of wounded cells, and 33% of cells isolated from the wound region. Na+ currents were also seen with a lower frequency in cells from the wound area. CONCLUSION: The majority of keratocytes migrating into a corneal freeze wound lose the voltage-gated K+ and Na+ ion channels present in cells from normal corneas. Ion channels from cells surrounding the wound site are minimally affected by the injury.

Characterization of voltage-gated, whole-cell ionic currents from conjunctival epithelial cells.

PURPOSE: These studies were performed to characterize the voltage-gated, whole-cell ionic currents in rabbit bulbar conjunctival epithelial and goblet cells. METHODS: New Zealand White rabbits were killed, and the bulbar conjunctiva was isolated. Conjunctival cells were dissociated for patch clamp analysis of whole-cell currents. The amphotericin, perforated-patch, whole-cell technique was used. RESULTS: Conjunctival epithelial cells had a mean capacitance of 6.72 pF (SE = 0.49; n = 25). The primary currents found were an inwardly rectifying K+ current, a saturating K+ current, and an outwardly rectifying nonselective cation current. A second nonselective cation current also appeared to be present. The inward current was observed in a KCl Ringer's bath and was almost nonexistent in a NaCl bath. The current was Ba(2+)- and Cs(+)-sensitive. The second K+ current became saturated at depolarized voltages and was Ba(2+)- and quinidine-sensitive. The first outward nonselective cation current was typically less than 100 pA in amplitude and activated at voltages positive to 0 mV. Tail current experiments showed that the current was cation selective. The current was blocked by Gd3+ but not by the Cl- current blockers 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid or 5-nitro-2-(3-phenylpropylamino)benzoic acid. The second nonselective cation current was larger and Gd(3+)-insensitive. The primary current observed in goblet cells was a large outward K+ current. CONCLUSIONS: The primary currents observed during whole-cell patch clamping of bulbar conjunctival epithelium are a Ba(2+)- and Cs(+)-sensitive, inwardly rectifying K+ current, a saturating K+ current, and two outwardly rectifying nonselective cation currents. Goblet cells contain a large outward K+ current.

Loss of fenamate-activated K+ current from epithelial cells during corneal wound healing.

PURPOSE: The corneal epithelium provides a barrier between the external environment and the cornea. It also serves as an ion transporting epithelium. Because of its proximity with the external environment, the corneal epithelium is frequently injured through physical or chemical insult. The purpose of this study was to determine whether corneal epithelial cell whole-cell currents change during corneal wound healing as the author of the present study has previously reported for corneal keratocytes and endothelial cells. METHODS: Rabbit corneal epithelial cells were injured by scraping, heptanol exposure, or freezing. The epithelium was allowed to heal for 12 to 74 hours. Cells were dissociated from corneas, and whole-cell currents were examined using the amphotericin-perforated-patch technique. RESULTS: Cells from the wounded corneal groups had significantly increased capacitance values, indicating increased surface area compared with that of control cells. As previously reported, the primary control whole-cell current was a fenamate-activated K+ current. An inwardly rectifying K+ current and a Cl- current were also observed. In epithelial cells from heptanol-wounded corneas, these conductances were generally unchanged. In cells from scrape- and freeze-wounded corneas, however, the fenamate-activated current was absent or significantly attenuated. CONCLUSIONS: As they do in corneal keratocytes and endothelial cells, K+ channels disappear during some models of corneal epithelial wound healing. In addition, cell capacitance, a measurement of cell surface area, increases. These results suggest that substantial K+ channel activity is not required for in vivo epithelial cell proliferation during corneal wound healing.

Keratocyte gap junctional communication in normal and wounded rabbit corneas and human corneas.

PURPOSE: Several studies have indicated the anatomic and biochemical presence of gap junctions in corneal keratocytes. The current study was designed to demonstrate that these gap junctions are functional in rabbit and human corneal keratocytes. This study also examined dye coupling between keratocytes migrating into the wound region of freeze-wounded rabbit corneas. METHODS: Freeze wounds were created on anesthetized rabbit corneas using a liquid nitrogen-cooled brass probe. Freeze-wounded corneas were examined at several time periods from days 0 to 5 after wounding. Nonwounded rabbit corneas also were examined. Human corneal buttons were examined immediately after removal from patients who underwent keratoplasty. Gap junctional coupling was examined by microinjecting carboxyfluorescein from microelectrodes into the basal-most keratocytes and capturing dye spread images with a cooled charge coupled device camera. RESULTS: Significant dye spread was observed between cells in the unwounded areas of corneas at wound time 0 and between cells migrating into the wound areas as early as 24 hours after wounding. In control corneas, dye spread to as many as 50 cells from the source cell. Dye spread also was seen between keratocytes in human corneas with pseudophakic bullous keratopathy and keratoconus. CONCLUSIONS: Gap junctions observed in keratocytes from normal rabbit corneas are functional. Gap junctions also are present and functional in keratocytes within unwounded and wounded regions of freeze-injured corneas. In addition, functional gap junctions are present between keratocytes in human corneas. This study confirms the long-held contention that corneal keratocytes form a large intercommunicating network within the corneal stroma.

Resting voltage measurements of the rabbit corneal endothelium using patch-current clamp techniques.

The resting potential (Em) of freshly isolated rabbit corneal endothelium was measured at room temperature (22 degrees C) and at 34 degrees C. Due to the wide range of values reported in the literature and the difficulty in obtaining long-term measurements using microelectrodes in these cells, a current-clamp technique was employed using whole cell patch-clamp electrodes. The electrodes contained a K+ methanesulfonate-based intracellular solution, and a NaCl/HCO3- Ringer's solution was used extracellularly. Three preparations of endothelium were examined: single dissociated cells, the isolated monolayer (stripped from the stroma with Descemet's membrane), and the intact isolated cornea. The perforated-patch technique, with amphotericin B in the electrode, was also used with the intact-cornea preparation at 34 degrees C. The mean Em values for the combined preparations at 22 degrees C and 34 degrees C were -35.3 mV and -55.0 mV, respectively; those for the intact-cornea preparation were -34.4 mV and -61.6 mV (at 22 degrees C and 34 degrees C, respectively). The isolated monolayer preparation showed a small but significant depolarization at both temperatures. These results demonstrate temperature dependence for Em in the corneal endothelium and show that more extensively dissected preparations have similar although not identical Ems to those of the intact cornea.

Phorbol ester modulation of rabbit corneal endothelial permeability.

PURPOSE: Phorbol esters have been shown to have a profound influence on cellular activity in many cell types. The purpose of this study was to examine the influence of phorbol esters on the function and structure of corneal endothelial cells. METHODS: Corneas were placed under a specular microscope, and the endothelium was superfused with glutathione bicarbonate Ringer's solution (GBR); with GBR and 10 nM, 100 nM, or 1 microM 4 beta-phorbol 12-myristate 13-acetate (PMA); or with 100 nM 4-alpha-PMA. Corneal swelling curves were generated, and endothelial permeability was determined. Corneal endothelial structure was examined with a scanning electron microscope. RESULTS: Significant increases in swelling and endothelial permeability were found in corneas perfused with 100 nM PMA versus that observed in controls (swelling rate = 26 microns/hr versus 6.9 microns/hr; permeability = 6 x 10(-4) cm/min versus 3.4 x 10(-4) cm/min) and in corneas receiving 1 microM PMA versus that in controls (swelling rate = 26.3 microns/hr versus 0.12 micron/hr; permeability = 6.9 x 10(-4) cm/min versus 4.9 x 10(-4) cm/min). Application of 10 nM PMA did not significantly alter either parameter. Study with transmission electron microscope demonstrated significant morphologic changes in cells perfused with all concentrations of PMA. Corneas perfused with 100 nM 4-alpha-PMA versus 100 nM PMA had significantly lower slope and permeability values (swelling rate = 5.9 microns/hr versus 25.1 microns/hr; permeability = 3 x 10(-4) cm/min versus 6.7 x 10(-4) cm/min). CONCLUSIONS: Phorbol esters are detrimental for corneal endothelial function, creating significant corneal swelling, increases in endothelial permeability, and changes in endothelial cell structure. This effect appears to be mediated through a protein kinase C pathway.

Properties of whole-cell ionic currents in cultured human corneal epithelial cells.

PURPOSE: To identify and partially characterize the ionic currents contributing to the whole-cell conductance of cultured human corneal epithelial cells. METHODS: Epithelial cells were scraped from human donor corneas and cultured for use in patch-clamp experiments. Amphotericin B and the perforated-patch configuration were used to measure whole-cell currents in cells isolated from confluent monolayers. RESULTS: Cell monolayers exhibited cobblestone morphology and were immunopositive for corneal epithelium-specific cytokeratin. Single cells had a capacitance of 21 +/- 2 pF and expressed similar types of ionic currents regardless of passage number. In descending order of frequency of occurrence, cells exhibited a nonselective cation current active at depolarized voltages and insensitive to Ba2+ and Gd3+; an outwardly rectifying K+ current active at depolarized voltages, stimulated by flufenamic acid and inhibited by tetraethylammonium; a voltage-gated inward Na+ current; an outwardly rectifying K+ current active at hyperpolarized voltages, stimulated by flufenamic acid, blocked by Ba2+, and insensitive to diltiazem; an inwardly rectifying K+ current; and a nonselective cation current inhibited by flufenamic acid. CONCLUSIONS: Our results are consistent with those in previous studies of noncultured epithelia from rabbit and human corneas showing an outwardly rectifying K+ current active at hyperpolarized voltages and a nonselective cation current active at depolarized voltages and insensitive to Ba2+. These data suggest cultured cells may be useful in determining the physiological role of ion channels in corneal epithelia and may aid in the development of a cell-based model for the examination of the effects of wounding and toxic agents on the human cornea.

Effect of tumor necrosis factor alpha on rabbit corneal endothelial permeability.

PURPOSE: Tumor necrosis factor alpha (TNF alpha) is present in the iris and the lacrimal gland, and its concentration is increased during inflammation and after corneal wounding. Although TNF alpha has been shown to increase keratocyte and corneal epithelial interleukin production, no definitive effects of TNF alpha on corneal endothelial cells have been reported. TNF alpha has been shown to disrupt barrier function in vascular endothelial monolayers through f-actin depolymerization. A reduction in intracellular cyclic adenosine monophosphate (cAMP) concentration may play a role in this response. This study was designed to examine the role and signal transduction mechanisms of TNF alpha modulation of endothelial permeability in the cornea. In addition, it is the first examination of the effects of TNF alpha on the barrier function of a noncultured cell monolayer. METHODS: Rabbit corneal endothelial superfusions were performed under an in vitro specular microscope. Corneas were processed for permeability measurements or f-actin staining. RESULTS: TNF alpha superfused corneas had significantly higher permeabilities than controls. f-actin staining revealed that TNF alpha superfusion disrupted f-actin filaments when compared to controls. Corneas superfused with the f-actin stabilizing agent phallacidin had significantly lower permeabilities than TNF alpha superfused pairs. Permeabilities of corneas superfused with TNF alpha plus 8-bromo-cAMP (0.01 to 3 mM) were significantly lower than TNF alpha superfused pairs at all concentrations, although only significantly lower at the 0.1 mM cAMP concentration. CONCLUSIONS: TNF alpha causes an increase in corneal endothelial permeability, and this increase is mediated by disruption of f-actin filaments; cAMP appears to be involved in this response.

Predicting refractive alterations with hydrogel keratophakia.

Hydrogel lenticules are being used as intracorneal lens (ICL) implants for refractive keratoplasty. The experimental surgical success can be evaluated through an understanding of their effect on the optical system of the cornea. An algorithm that utilizes elementary optics can be used to calculate the total corneal power produced through intracorneal lens implantation via either pocket or microkeratome dissection. Two groups of animal experiments involving ICL implantation using both pocket and microkeratome dissections were performed on Rhesus monkeys. The predicted effects of the surgical techniques were compared with the measured effects obtained via streak retinoscopy. The mean difference and standard deviation of the measured minus the predicted values for the pocket dissection group is -0.59 +/- 1.52 D, N = 7. The respective difference for the microkeratome group is -0.19 +/- 1.07 D, N = 4. These small differences illustrate the accuracy of the algorithm in predicting the effects of refractive keratoplasty with hydrogel intracorneal lenses.

Corneal endothelial junctions and the effect of ouabain.

Paired rabbit corneas were perfused in vitro for endothelial permeability (Pac) determination with glutathione bicarbonate Ringer's solution (GBR) and GBR plus ouabain (10(-4) M). Results indicated no difference in Pac between the two groups (3.39 vs 3.67, respectively) despite significantly greater stromal swelling in the group perfused with ouabain. Freeze-fracture microscopy of similarly perfused corneas revealed intact tight junctional complexes in both groups, although the tight junctional complex of perfused corneas appeared less organized than that of freshly enucleated, nonperfused controls. Gap junctions were abundant as observed in freeze-fracture replicas of GBR-perfused endothelium, and appeared to be decreased or absent in ouabain-perfused endothelium. These results indicate that corneal endothelial tight junctions are unaffected by perfusion with ouabain, whereas gap junctions appear to be lost. The permeability and freeze-fracture data reaffirms the importance of tight junctions as permeability barriers and indicates that gap junctions are not of primary importance for maintenance or control of the corneal endothelial barrier.

Phospholipid growth factors and corneal wound healing.

In many tissue types, wound healing involves cell division and migration over and into the wound area to cover and remodel the wound. LPA and other members of the phospholipid lipid growth factor (PLGF) family stimulate many of the activities involved in wound healing. In the rabbit cornea, we have found that keratocytes from wounded corneas have a volume-activated Cl- current activated by LPA and alkenyl-LPA. This current is minimally activated by cyclic PA and SPC, and is not activated by LPA in cells from uninjured corneas. Biochemical examination of PLGFs in aqueous humor and lacrimal fluid before and after wounding identified LPA, alkenyl-GP, PA, and lyso PS, with elevated PLGF activity after wounding. In recent experiments examining human corneal cell lines and cultured cells using RT-PCR, we found mRNA for EDG receptors 1-5, with an apparent increase in EDG-3, -4, and -5 following brief SDS application to cell lines, and EDG receptors 2-5 induction in late-passage human corneal epithelial cells. This work points to a significant role for PLGFs in the corneal wound-healing process.

Human corneal storage in modified McCarey-Kaufman and K-Sol media: effect on endothelial Na+/K+ ATPase pump site density and permeability.

Endothelial permeability (Pac) to carboxyfluorescein and Na+/K+ ATPase pump site density were determined in human corneas following storage for 4 or 7 days at 4 degrees C in either modified McCarey-Kaufman (mMK) or K-Sol media. Following 4 days of storage, Pac values for mMK- and K-Sol-preserved corneas were not significantly different from those of their prestorage mates. After 7 days of storage, however, corneas stored in K-Sol media showed a significant increase in Pac compared to their prestorage mates, whereas the mMK-stored corneas showed no change in Pac. Na+/K+ ATPase pump site density determined using [3H]ouabain was similar to a control group in the K-Sol-stored tissue but higher in the mMK-stored tissue following 7 days of storage. These studies suggest that mMK medium maintains endothelial barrier function and Na+/K+ ATPase pump site density at least as well as K-Sol medium through 7 days of corneal storage.

Initial characterization of whole-cell currents from freshly dissociated corneal keratocytes.

The perforated patch technique was utilized to obtain whole-cell currents from freshly dissociated rabbit corneal keratocytes. We describe and provide the initial characterization of two distinct whole cell currents in rabbit keratocytes: a K(+)-selective delayed rectifier and a voltage-sensitive, tetrodotoxin blockable Na+ current. The voltage-sensitive Na+ current is of sufficient magnitude to allow us to initiate action potentials when current-clamping the cells. This is the first detailed electrophysiological study of corneal keratocytes.

Dye spread through gap junctions in the corneal epithelium of the rabbit.

PURPOSE: Microelectrode dye injection of 5,6-carboxyfluorescein was used to investigate gap junctional communication in the corneal epithelium. METHODS: Dye injection started in the superficial layer and proceeded stepwise into the underlying epithelial layers until spread was observed. Intracellular [Ca2+] was manipulated by exposing the cornea to the calcium ionophore A23187 (global increase) or by increasing the [Ca2+] in the injection electrode (source cell increase). Intracellular pH was manipulated by exposing the cornea to nigericin in a low-pH KCI Ringer's (global decrease) or by lowering the pH in the injection electrode (source cell decrease). Heptanol was tested for its ability to uncouple gap junctions. Gap junctional communication was based on the layer at which spread was first observed and on the apparent dye travel distance from the point of injection. RESULTS: Control dye spread occurred, on average, in the third layer from the surface. Increased [Ca2+] in the source cell resulted in an initial spread occurring in the second layer. Globally increasing [Ca2+] with A23187 resulted in no change in the average initial spread layer. Lowering intracellular pH of the source cell did not affect the initial dye spread layer. Globally lowering intracellular pH resulted in significant gap junctional inhibition in a time-dependent manner. Dye spread distance was not significantly affected by [Ca2+] or pH manipulations. Heptanol (2.5 mM) completely inhibited dye coupling. CONCLUSION: All cell layers of the corneal epithelium contain functional gap junctions, although it appears that intercellular communication in the superficial layers does not occur under our control conditions. Intercellular communication through these junctions can be altered by various manipulations of [Ca2+] and pH.

Comparison of conjunctival and corneal surface areas in rabbit and human.

This comparative study shows the surface area ratio of conjunctiva to cornea to be two times larger in humans than in rabbits. This large heretofore unrecognized interspecies difference may affect the applicability of drug pharmacokinetic data obtained using rabbit models and should be taken into consideration in topical drug development and future comparative drug penetration studies between rabbit and man.

A method for the in vitro determination of feline corneal endothelial permeability.

A mounting block for in vitro perfusion of the cat cornea is described. Using this apparatus and the techniques of Araie, the permeability (Pac) of the normal cat corneal endothelium to carboxyfluorescein was determined to be 2.5 +/- 0.2 x 10(-4) cm/min. To assess the sensitivity of this technique in determining changes in Pac associated with alterations in endothelial, morphology, three cats underwent 2 successive unilateral, central, 10 mm diameter circular areas of endothelial debridement 6 weeks apart. Six weeks following the second wounding all 3 animals underwent morphometric analysis and Pac determination. A trend toward an elevation in Pac with extreme reductions in cell density was observed.

A collagen-based scaffold for a tissue engineered human cornea: physical and physiological properties.

Stabilized collagen-glycosaminoglycan scaffolds for tissue engineered human corneas were characterized. Hydrated matrices were constructed by blending type I collagen with chondroitin sulphates (CS), with glutaraldehyde crosslinking. A corneal keratocyte cell line was added to the scaffolds with or without corneal epithelial and endothelial cells. Constructs were grown with or without ascorbic acid. Wound-healing was evaluated in chemical-treated constructs. Native, noncrosslinked gels were soft with limited longevity. Crosslinking strengthened the matrix yet permitted cell growth. CS addition increased transparency. Keratocytes grown within the matrix had higher frequencies of K+ channel expression than keratocytes grown on plastic. Ascorbic acid increased uncrosslinked matrix degradation in the presence of keratocytes, while it enhanced keratocyte growth and endogenous collagen synthesis in crosslinked matrices. Wounded constructs showed recovery from exposure to chemical irritants. In conclusion, this study demonstrates that our engineered, stabilized matrix is well-suited to function as an in vitro corneal stroma.

Properties of porcine and recombinant human collagen matrices for optically clear tissue engineering applications.

Porcine and recombinant human atelocollagen I solutions were cross-linked with a water soluble carbodiimide at various stoichiometries and collagen concentrations (5-20 w/w %). The resulting hydrogels were clear and, when used as cell growth matrices, allowed cell and nerve visualization in vitro and in vivo. We have previously reported that, after six months of implantation in pigs' and rabbits' corneas, these robust hydrogels allowed regeneration of host cells and nerves to give optically clear corneas with no detected loss in thickness, indicating stable engraftment. Here, the biocompatible hydrogel formulations leading to this novel in vivo performance were characterized for amine consumption, gel hydration, thermal properties, optical clarity, refractive index, nutrient diffusion, biodegradation, tensile measurements, and average pore diameters. Gels with excellent in vitro (epithelial overgrowth, neurite penetration) and in vivo performance (clarity, touch sensitivity regeneration) had 4-11 nm pores, yet had glucose and albumin diffusive coefficients similar to mammalian corneas and allowed neurite extension through the gels.