Isolated Rabbit Corneas Research Articles

The Origins of Sattler's Veil

The symptoms of Sattler's veil accompany prolonged wear of thick contact lenses and are thought to be the result of epithelial hypoxia. We studied light scattering in the isolated rabbit cornea after hypoxia with optical techniques. We found that hypoxia produced a polygonal mesh of light scattering, outlining cells in the intermediate and basal epithelium. An optical transform of this mesh produced a halo of the same dimensions observed in Sattler's veil. Additionally, prolonged hypoxia produced an increase in stromal light scattering that may add to increased glare sensitivity. Consistent changes in epithelial thickness were not observed, raising the probability of radial swelling or compensatory epithelial volume regulation.

The penetration of exogenous prostaglandin and arachidonic acid into, and their distribution within, the mammalian eye.

The distribution of 3H and 14C activities in intraocular fluids and tissues was studied after topical application of 3H-prostaglandin F2 alpha (3H-PGF2 alpha) and/or 14C-arachidonic acid (14C-AA) to rabbit eyes; intravenous (i.v.) infusion of 3H-PGF2 alpha into rats; and after incubation of rat or rabbit globes in media containing these and other tracers. Thirty min after topical application of 14C-AA, the order of distribution of 14C per unit of tissue weight was cornea much greater than sclera congruent to ciliary body congruent to iris congruent to aqueous greater than retina-choroid greater than vitreous greater than lens. The distribution of 3H was sclera greater than cornea greater than retina-choroid congruent to ciliary body greater than aqueous greater than vitreous (lens congruent to 0; iris congruent to 0). After i.v. infusion of 14C-sucrose and 3H-PGF2 alpha into rats for 1 to 5 min, the globe/blood ratio of 3H was significantly lower than that of 14C. When isolated rat globes were incubated, the order of tracer uptake into the whole globe was AA greater than thiourea greater than PGF2 alpha greater than sucrose, while the order of entry into the aqueous was thiourea greater than sucrose greater than AA greater than -PGF2 alpha. The isolated rabbit cornea accumulated large amounts of 14C-AA which was not readily elutable and much smaller amounts of 3H-PGF2 alpha which was readily elutable. It is concluded that the sclera is highly permeable to PGF2 alpha, but that the cornea is an effective permeability barrier to this, and presumably all other PGs. The passage of AA through the outer coats of the globe is limited by its chemical incorporation into the cornea, sclera and conjunctiva. Thus, inhibition of the adverse intraocular effects of topically applied AA by topically applied drugs may only reflect the ability of these drugs to inhibit the synthesizing capacity of the superficial layers of the globe rather than that of the anterior uvea and other intraocular tissues.

Influence of Cetylpyridinium Chloride on Corneal Permeability to Penicillin

The epithelial surface or the deepithelialized anterior stromal surface of isolated rabbit corneas was perfused for 3 hr with14C‐penicillin in 25 mMRinger‐bicarbonate solution with or without 1% albumin and with or without 0.02% cetylpyridinium chloride. The intact epithelium acted as a barrier to penicillin and impeded the flux rate by 66% when compared to the flux rate across the deepithelialized cornea. The presence of 0.02% cetylpyridinium chloride increased the penicillin flux rate across corneas with an intact epithelial layer to that of deepithelialized corneas. Cetylpyridinium chloride, 0.02%, had no effect on penicillin flux across deepithelialized corneas. The penicillin flux rate across corneas, with or without epithelium, was increased slightly following the inclusion of 1.0% albumin in the bathing solution. The flux rates across deepithelialized corneas in the presence of albumin, with or without cetylpyridinium chloride, were similar to fluxes found in the absence of albumin. Albumin‐penicillin “binding” was not a significant factor in impeding penicillin flux, and this binding apparently was not altered by cetylpyridinium chloride. The surfactant appeared to alter epithelial permeability physiologically.

Intracellular potentials of isolated rabbit and human corneal endothelium

Cell membrane potentials of corneal endothelium were studied with 3 m-KCl-filled micro-electrodes using the glass fibre method of filling. Cell potential measurements were accepted when stable for at least 2 sec. In the isolated rabbit cornea a mean potential of 30·6±1·2 mV (range 7–93 mV) was recorded up to 5 hr after incubation. In some experiments potentials of about 60 mV and stable for several minutes could be obtained. Potentials were depolarized to 13·8±0·6 mV by 150 m m-KCl and 15·6±0·8 mV by 10 −3 m-NaCN and abolished by seraping off the endothelium. Endothelial potential in isolated human corneas of enucleated eyes (with normal corneal function) was 19·9±4·4 mV. A mean of 18·1±1·2 mV was obtained in cadaver corneas taken 2–5 hr after death. Cell potential decreased to 12·5±0·8 mV when corneas were stored at 4°C for 12–18 hr before incubation in Ringer's solution.

Intracellular potentials of isolated rabbit and human corneal epithelium

Cell membrane potentials (p.d.) of corneal epithelium were studied with 3 m-KCl-filled microelectrodes using the glass fibre method of filling. In the isolated rabbit cornea a staircase-like potential profile with five to seven single steps was obtained and a mean intracellular p.d. of 68·5±0·6 mV was recorded in basal epithelial cells. This p.d. was stimulated to 78·2±0·7 mV by 3·7 m m ascorbic acid and inhibited by KCl and NaCN. Epithelial p.d. in isolated human corneas of enucleated eyes (with normal corneal function) was 57·4± 0·4 mV. Cadaver corneas removed 2–5 hr after death or additionally stored for 12–18 hr in a moist chamber at 4°C exhibited, at least for 2 hr of incubation in modified Ringer solution, an adequate epithelial cell potential.

Rabbit endothelial response to ophthalmic preservatives.

The endothelial surface of isolated rabbit corneas were perfused for three hours with varying concentrations of benzalkonium chloride and cetylpyridinium chloride. The threshold for physiological and ultrastructural alteration of corneal endothelium is approximately 0.0001% for benzalkonium chloride and 0.01 mM for cetylpyridinium chloride. The effects of the surfactants can be induced with as little as a 15-minute exposure without subsequent recovery. Use of ophthalmic medications or irrigating solutions containing these agents inside the eye is potentially hazardous to the corneal endothelium. Topical administration of 0.133% benzalkonium chloride to the anterior surface of deepithelialized in vivo corneas (five doses, seven minutes apart) caused no alterations of corneal endothelial cell function or ultrastructure.

A study of the transfer of amino acids across the endothelium of the rabbit cornea

The purpose of this study was to determine whether the endothelium of the cornea is capable of transporting amino acids from the aqueous humor to the extracellular fluid of the stroma against a concentration gradient. Isolated rabbit corneas, free of epithelium, were placed in a chamber and perfused independently on both surfaces with an artificial aqueous humor. The flux of 14C-labelled amino acids across the tissue was then determined both from the aqueous to tear side and from tear side to aqueous. The permeability of the endothelium to each amino acid was found to be equal in the two directions. It is concluded that movement against a concentration gradient from aqueous to stroma is, therefore, not possible. These findings are consistent with measured levels of amino acids in the cornea in vivo, and indicate that stromal concentrations higher than those in the aqueous humor are the result of accumulation in the keratocytes rather than of active transport across the endothelium. The boundary cell layer of the cornea, unlike that of the lens, appears to have no active nutritive role in the supply of amino acids from the aqueous humor.

Transport of Na, Cl, and water by the rabbit corneal epithelium at resting potential

Theophylline (1 mM) produced a net transport of Na and Cl from aqueous humor to tears (.02-.04 mumol/cm2 h) in the isolated rabbit cornea denuded of endothlium and in the presence of normal resting potential (25-35 mV). The active transport of Na (tears to aqueous) and of Cl (aqueous to tears), estimated with the Goldman constant-field equations, was confirmed. A 10 degrees C rise in temperature produced changes close to those predicted for passive processes in both unidirectional fluxes of Na and in the tears-to-aqueous flux of Cl, but not for the aqueous-to-tears flux of Cl. Theophylline treatment doubled Cl permeability but did not significantly affect Na or urea permeability, suggestingspecificity of affect. In separate experiments it was shown that stromal thinning occurred in previously swollen corneas when the endothelium was blocked by silicone oil and the epithelium was treated with theophylline. These findings provide further support for the argument that the mammalian epithelium could have an active role in the regulation of corneal thickness in situ.

Sodium and chloride transport across the isolated rabbit cornea

Sodium and chloride unidirectional fluxes through rabbit corneas in vitro have been measured. The natural cornea curvature was simulated by applying a hydrostatic pressure on the endothelial side. Fluxes were correlated to spontaneous potential differences and short-circuit currents in order to determine the components of passive and active transports. There is active transport of sodium toward the aqueous humour. Chloride ions are actively transported toward the tears. Both contributions do not add up to the short-circuit current. A tentative explanation in terms of non-steady-state conditions is proposed.

Mass Transfer in the Cornea: II. Ion Transport and Electrical Properties of a Series Membrane Tissue

The electrical and active transport properties of isolated rabbit cornea are investigated by computer experimentation. The tissue is modeled as a series membrane system and the passive ion fluxes through it are described by the frictional formulation of irreversible thermodynamics. From short-circuit current (SCC) data, it is found that the epithelial sodium pump rate (P) is not appreciably changed when much of the sodium in the solution bathing the anterior corneal surface (concentration = c(11)) is replaced by choline, with choline-free medium posteriorly. Simulations of open-circuited corneas, using the mean P computed from the SCC data, yield corneal and stromal potentials in agreement with experiment. The stromal fluid is calculated to become more hypotonic as c(11) is diminished, a result consistent with posttest measurements of the sodium content of experimental stromata. The apparent decrease in "bound sodium" which accompanies the reduction of c(11) is a result of the associated changes in steady stromal hydration; the epithelial sodium pump does not contribute to corneal deturgescence. The inclusion of a simple epithelial structure in the computations changes the value of P but affects neither its constancy nor the calculated behavior of the cornea under open-circuit conditions. A general algebraic relation among pump rates and ion fluxes in short-circuited series membrane systems bathed in complex media is derived and used to construct a relation between P and SCC for the cornea. This equation yields pump rates in good agreement with the computer results and is used to show that (a) P is independent of c(11) if d(SCC)/dc(11) is a constant related to the over-all corneal permeability to sodium, and (b) a Lineweaver-Burke plot of 1/SCC vs. 1/c(11) can appear to be linear at constant P.

Ion transport across the isolated rabbit cornea

Ion flux determinations, made during the first hour after excision of the cornea from the rabbit, have shown that both sodium and chloride ions are actively transported across the cornea from the epithelial to the endothelial surface. The sodium transport persists for several hours, while the chloride movement is of a purely passive nature 1 hr after removal from the animal. This finding is supported by measurements of the electrical parameters of the membrane made in media of different natures. Bathing the tissue in normal Ringer's solution gives a potential difference of 5–6 mV, in choline Ringer's solution the potential difference becomes negative by −2 to −3 mV, while in sulfate Ringer's solution the potential difference rises to maxima of 15–18 mV. The location of a transport “pump” is suggested to be in the epithelium.

Ion transport in isolated cornea of the rabbit.

When the isolated rabbit cornea is bathed with well-stirred normal Ringer solution, only a low potential difference (PD) exists across the tissue; the initial value of 2 mv rises to 6 mv (endothelium positive) 1 hr after excision from the animal. In sodium-free Ringer solution the PD becomes negative before becoming negligible, while in chloride-free Ringer the PD rises to triple the value in normal Ringer. Flux measurements of sodium 22 show that there is an initial inequality between the net flux and the measured short-circuit current (SCC), the values of which become equal 1 hr after removal of the cornea from the animal Flux measurements of chloride 36 during this 1st hr indicate an active transport of chloride inward across the cornea, but after 1 hr the fluxes are equal in each direction. The differences of the net currents generated by the sodium and chloride transports equal the measured SCC, and the two transports have been shown to be able to exist independently.