David M. Maurice

A Simple Conjunctival Biopsy

Biopsy of conjunctival goblet cells may be carried out by pressing a piece of Millipore filter on the conjunctival surface. This can then be stained with PAS and hematoxylin and observed under the microscope after the filter has been cleared with oil. Either spots of mucous corresponding to the openings of the goblet cells are seen, or the filter adheres to the surface and pulls off one or more layers of epithelial cells and goblet cells. The procedure can be carried out on the unanesthetized eye and does not cause discomfort.

The mechanical properties of the rabbit and human cornea

The extensibility of rabbit and human corneas was measured by raising the pressure within the intact globe of the eye and measuring the displacements of two very small mercury drops on the corneal surface. The human cornea showed a negligible extensibility under low stresses. The rabbit tissue, however, underwent a 9% strain under low pressures with a curvilinear relationship between stress and strain. At higher pressures the relationship was linear, and the tissue showed some creep. The low pressure stress-strain relationship of the rabbit could not be explained on the basis that the collagen fibrils were being straightened out from an initial set in a sinusoidal wave. When the stroma was isolated from Descemets membrane, it showed a negligible low pressure extensibility in rabbit and man. On the other hand, isolated Descemets membrane was very extensible in both species. The difference between them in the behavior of the intact cornea seems to lie in the relative initial strain in the stroma and Descemets membrane.

Diffusion across the sclera.

Abstract Regions of beef sclera free from perforations were mounted between two chambers and the movement of substances from one side to the other determined. All dyestuffs tested were virtually impermeant except for fluorescein and acid fuchsin. A variety of ions and solutes up to the size of serum albumin were also tested and were found to diffuse across about three times more slowly than they do in the rabbit corneal stroma. This rate of diffusion would slow the passage of locally applied drugs through the selera to an extent compatible with the delay in their action on the pupil.

Corneal endothelial transplantation.

Patients with visually significant corneal edema, secondary to endothelial dysfunction, before the development of scaring or vascularization, need only have the corneal endothelium replaced to restore corneal clarity. This fact, plus the lack of consistently available donor material, prompted us to evaluate tissue cultured corneal endothelim (TCCE) as a donor source. We have shown that TCCE, when transplanted, can regain normal morphology and physiologic function. To accomplish practical use of autologous stroma, a transparent gelatin membrane which can serve as substrate for endothelial growth in tissue culture, has been developed. This cellular membrane has been transplanted successfully in rabbits with good functional results. It is hoped that ultimately this technique can be developed for routine use in man.

The loss of fluorescein, fluorescein glucuronide and fluorescein isothiocyanate dextran from the vitreous by the anterior and retinal pathways

The pathways by which fluorescein (F), fluorescein glucuronide (FG) and fluorescein dextran (FD) leave the vitreous body of the rabbit were examined by measuring the concentration distribution of the injected fluorophores in sections of the frozen eyes. The contours of F, as already known, show that it leaves the vitreous predominantly across the retinal surface. Mathematical analysis of the concentration gradient leads to an average outward permeability coefficient of 1.4 x 10(-3) cm min-1 for the retinal layers. The contours of FG and FD show that they leave predominantly by diffusion into the posterior chamber, encountering only a minor barrier at the anterior hyaloid membrane. The anterior contours indicate that there can be no substantial posteriorly directed fluid flow through the vitreous; if it occurs its velocity across the retinal surface must be less than 2 x 10(-5) cm min-1. The contours of FD near the posterior pole of the retina suggest that such a flow may be taking place. Some time after the systemic administration of F, an analysis of the rate of loss of fluorescence from the vitreous body shows that this corresponds to the movement of FG out through the anterior chamber. Its value bears little relationship to the condition of the blood-vitreal barrier.

Transplantation of tissue-cultured corneal endothelium

Cultured endothelial cells have been shown to regain their physiological function when replaced in the rabbit eye. Corneas were wiped free of native endothelium and seeded with cultured cells. After an incubation period, full-thickness buttons were cut from these corneas and transplanted into recipient animals. Clear grafts were obtained only when the donor cells were derived from cultures less than a month old. Light and scanning electron microscopy showed the endothelial cells of these grafts to be present as a slightly irregular monolayer on the posterior surface of the cornea. In corneas made edematous by benzalkonium chloride, the clear graft remained surrounded by thick and cloudy host tissue. In those grafts with 3H-thymidine--labeled cells, radioactivity was limited to the host tissue.

Electrical potential and fluid transport across the corneal endothelium

Abstract The electrical potential and the fluid pump across the endothelium of the excised rabbit cornea were measured simultaneously. When the pump was operating at its maximum, the average potential was 1·3 mV, with the aqueous humor negative to the stroma. Factors that inhibited the pump also diminished the potential, but the drop in the latter usually preceded that in the former. It is calculated that if the potential results from an anion pump which transports water by means of an osmotic mechanism, then the ion flux will be barely adequate to account for the water movement. Three alternative mechanisms which could give rise to the potential but which would not be directly responsible for the fluid transport are discussed.

The rate of diffusion of fluorophores through the corneal epithelium and stroma.

The time taken to cross the rabbit corneal epithelium and stroma was estimated for fluorescein (F), carboxyfluorescein (CF), rhodamine B (RB), and sulforhodamine B (SRB). Paired corneas were mounted in vitro; one was intact and the dye solution was kept in continuous contact with its epithelial surface; the epithelium was scraped from the other and the dye was applied as a pulse to the bare stroma. The time course of the dye appearing in a solution rapidly passing over the endothelial surface was determined by fluorometry. This rate of appearance was compared in the two cases and used to estimate the diffusional lag time introduced by the epithelium. For the very hydrophilic CF and SRB, the delay was too short to measure; this is compatible with the passage of these dyes taking place through the paracellular spaces. For the very lipophilic RB, the delay was about 2 min; this was rather too slow for it to be explained as being controlled entirely by diffusion in the cytosol. For the intermediate F, the delay was 5 min; it is suggested that this is a result of it partitioning between the spaces and the cytosol during its passage. The experiments also led to determinations of the permeability of the epithelial and endothelial layers to the dyes. In both cases lipophilicity was a strong determinant of penetration, but not the only one. The permeability of the endothelium to F was unchanged from its in vivo value in these experiments, but that of the epithelium was increased four-fold. The diffusion rate of the dyes across the stroma could also be determined. There was no clear relationship with molecular size or partition coefficient. The rate of diffusion of F across the tissue was about half that in its plane, as determined in previous experiments. This is possibly a result of the anisotropic structure of the tissue.

Striations of light scattering in the corneal stroma.

When the rabbit cornea is stressed in various ways, light focused on the stroma is scattered back in the form of striations. Light and electron microscopy reveal that the striations correspond to a waveform assumed by the collagen fibrils in the stroma when the tension on them is relaxed. The waves act as reflectors for the light, which is a consequence of the light-scattering properties of the fibrils themselves, rather than a result of interfaces opening up between the lamellae.

Iontophoresis of Fluorescein into the Posterior Segment of the Rabbit Eye

Iontophoresis of appreciable quantities of fluorescein into the vitreous body of the rabbit results from the use of a high electrical current density over a limited area of the globe. This is achieved by passing current through the fluorescein when it is held against the region of the ora serrata in a tube less than 1 mm in diameter. The retina is destroyed over a corresponding area when the current enters the eye. An electrode is described which is held to the eye by suction and could be practical for clinical applications.