Comparison of rabbit and human corneas stored in Optisol-GS: changes in stromal sodium.

PURPOSE: To evaluate the hydration, and the levels of free, total and bound sodium in fresh rabbit corneal stromas and also those preserved for up to 21 days in Optisol-GS. The effect of epithelial removal on stromal sodium and hydration parameters was also evaluated. Trends in stromal hydration and sodium environment were compared to results we previously obtained using human eyes stored under identical conditions. METHODS: Stromal hydration was evaluated thermogravimetrically. A sodium-specific electrode and an atomic absorption spectrophotometer were used to determine the amounts of free and total stromal sodium, respectively. In one cornea of each pair, the epithelium was removed prior to placement in the storage media. After 3, 7, 14 or 21 days at 4 degrees C, corneas were removed from the Optisol-GS, at which time sodium and hydration measurements were obtained. RESULTS: With an intact epithelium, the hydration of the rabbit stromas was elevated significantly at each day of storage compared to fresh corneas. Free and total sodium levels of rabbit stromas did not differ statistically from fresh values, however the bound sodium values did increase during storage. In the absence of the epithelium, the stromal hydration and sodium content (free, total and bound) were significantly elevated and the increase was much greater than in corneas stored with an intact epithelium. These findings differ from those we measured previously using human tissue. CONCLUSIONS: Rabbit corneas responded differently from human corneas to storage in Optisol-GS. The hydration levels increased to a greater level in rabbit than human corneas under both storage conditions. The trends in amounts of both free and total sodium were similar between the species, although the absolute amounts differed. The largest discrepancy was observed in the amount of bound sodium, with the rabbit corneas experiencing large increases not documented in the human tissue. These results suggest that direct comparisons of stromal hydration and ionic environment between the species should be approached with caution.

Storage of human corneas in dextran and chondroitin sulfate-based corneal storage medium: changes in stromal free sodium.

OBJECTIVES: To evaluate the hydration and the levels of free and total sodium in human corneal stromata preserved for up to 21 days in a dextran and chondroitin sulfate-based corneal storage medium (Optisol-GS, Chiron IntraOptics, Irvine, Calif) and to evaluate the effect of epithelial removal on stromal sodium and hydration parameters. METHODS: Stromal hydration was evaluated thermogravimetrically. A sodium-specific electrode and an atomic absorption spectrophotometer were used to determine the amounts of free and of total stromal sodium, respectively, of preserved human corneas. In 50% of the corneas, the epithelium was removed prior to placement in the storage medium. After 3, 7, 14, or 21 days at 4 degrees C, corneas were removed from the storage medium and sodium measurements were taken. RESULTS: In corneas with an intact epithelium, the stromal hydration as well as the stromal free sodium and total sodium levels were relatively constant up to 21 days of storage in the preservation medium. In the absence of the epithelium, the water and sodium contents of the stroma increased significantly during storage. CONCLUSION: The presence of an intact epithelium is required for maintaining the hydration and sodium levels within the corneal stroma during storage. Removal of the epithelium prior to storage results in increased sodium values and hydration, which may affect postkeratoplasty deturgescence.

Three homologs of rds/peripherin in Xenopus laevis photoreceptors that exhibit covalent and non-covalent interactions.

We have isolated and characterized three homologs of mammalian rds/peripherin from Xenopus retinae. One (xrds38) is likely the Xenopus ortholog, while the other two (xrds36 and -35) are more distant relatives. By immunocytochemical analysis of retinal sections, xrds38 is distributed in both rod and cone photoreceptors, while xrds36 and xrds35 are present in rods only. At the EM level, xrds38 is present specifically in the rims and incisures of rod and cone outer segment discs. All are N-glycosylated and form covalent dimers. Immunoprecipitation analysis showed that in rods, these three proteins interact to form heterotetrameric or higher-order complexes. The pattern of sequence conservation among the xrds proteins, mammalian rds/peripherin, and mammalian rom-1 suggest that the central portion of the intradiscal D2 loop contains the interacting structural elements.

Cell birthdays in Xenopus laevis retina.

Using an in vitro differentiation system, we reevaluated the stages of development during which retinal neurons become postmitotic in Xenopus laevis. We also examined whether retinal detachment and removal of the pigment epithelium stimulated proliferation of previously postmitotic retinal cells. Retinas with and without an adherent pigment epithelium (stages 24-40 and stage 33/34, respectively) were removed from X. laevis embryos, placed in culture and allowed to differentiate in the presence of 3H-thymidine. After 2 days, eyes were fixed and processed for autoradiography. The proportion of labeled to unlabeled nuclei in the posterior pole of the retina was determined for each of the three cell layers. Early in development, most unlabeled cells were found in the ganglion cell layer; at stage 24, 53% of the cells showed no labeling, but by stage 32-33/34 all of the cells present were unlabeled. Within the outer nuclear layer, 17% of the cells failed to incorporate the 3H-thymidine at stage 24 and by stage 33/34, 100% of the cells were unlabeled. Within the inner nuclear layer, 13.5% of the cells failed to show labeling at stage 24, whereas at stage 40, none of the cells were labeled. There was no difference in the proportion of labeled to unlabeled nuclei in any of the cell layers when retinas were allowed to differentiate either with or without an adherent pigment epithelium. These results indicate that as early as stage 24, some cells that will become positioned in each of the nuclear strata fail to incorporate 3H-thymidine, suggesting that these cells become postmitotic very early in neurogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Outer segment disc membrane assembly in the absence of the pigment epithelium: the effect of exogenous sugars.

When retinal rudiments of Xenopus laevis embryos are allowed to differentiate in vitro, membrane precursors destined for the outer segment do not form well-ordered stacks of membrane leaflets if the pigment epithelium is removed from the outer retinal surface. However, addition of certain saccharides permits the organization of outer segment membranes into stacked membranous saccules, much like that demonstrated during in vivo or in vitro development in the presence of an investing pigment epithelium. Sugars that support the formation of well-organized photoreceptor outer segments include lactose, galactose and their derivatives, while numerous other carbohydrates failed to support outer segment organization.

Photoreceptor outer segment development in Xenopus laevis: influence of the pigment epithelium.

Opsin gene expression, synthesis, and photoreceptor outer segment morphology were evaluated during retinal development in Xenopus laevis. Retinal rudiments were harvested during in vivo development from embryonic stages 31 through 46 or were allowed to develop in vitro after removal from stage 33/34 embryos for 1, 2, or 3 days either with or without an investing pigment epithelium. Opsin mRNA was detected at stage 33/34 and the transcript level increased until stage 40 and remained at this level through stage 46. Opsin was first detected at stage 37/38 and progressively increased through stage 46. Rudimentary photoreceptor outer segment membranes occasionally appeared as early as stage 33/34 and they gradually increased in length, forming well-defined stacks of collapsed membranous saccules (discs) during in vivo development. The maturation of eye rudiments in culture was followed to determine how closely in vivo and in vitro development compare and to examine the ability of photoreceptors to differentiate when maintained in the absence of an overlying pigment epithelium (PE) layer. With the PE present, opsin mRNA as well as opsin content steadily increased over the entire culture period. After 1 day of culture, short cilia with minimal amounts of outer segment membranous material were present. By Day 3, the degree of outer segment differentiation corresponded morphologically to approximately stage 43 of in vivo development. When cultured in the absence of an investing PE, the opsin mRNA level increased minimally during the 3 days in culture. Opsin content increased, yet the relative amount was approximately 50% less than that present in retinas developing in the presence of the PE. Membranous material was elaborated; however, the outer segments appeared to be highly disorganized and formed whorl-like structures rather than the normal stacked disc morphology. These results suggest that the PE may be involved in regulating opsin at the transcriptional and/or translational levels and also participates in the organization of rod outer segment membranes.

Sodium activity in the aqueous humor and corneal stroma of the rabbit.

The present study examined the free sodium concentration of the aqueous humor and corneal stroma of both transparent and non-transparent corneas to assess the transendothelial activity gradient for sodium. In the transparent cornea of the adult rabbit, the sodium activity was higher in the aqueous humor than the stroma. This difference in sodium activity would cause water to diffuse down its concentration gradient from stroma to aqueous humor. In this way corneal transparency and the deturgesced state are maintained. Removal of the corneal endothelium in the adult rabbit produced an opaque swollen cornea. Under these conditions the sodium activity was higher in the stroma than the aqueous humor. However, an osmotic gradient was not produced by the Na+ activity gradient because the endothelium was not present to act as a semi-permeable membrane. The corneal endothelium was no longer present to establish and sustain the activity gradient for sodium that is necessary for corneal transparency in the mature rabbit. The transendothelial sodium activity gradient was also measured in 13-day-old rabbits. At this age, the cornea was not yet transparent, nevertheless the free sodium concentration of the aqueous humor was higher than that of the stroma, similar to the adult transparent cornea. This suggests that forces other than the establishment of the proper transendothelial sodium gradient are responsible for the lack of corneal transparency in the young rabbit.

Maturation of the corneal endothelial tight junction.

Apical tight junctional formation of the rabbit corneal endothelium was examined by freeze-fracture analysis and measurement of paracellular permeability to 5(6)-carboxyfluorescein. Freeze-fracture analysis indicated that apical tight junction formation of the rabbit corneal endothelium is a dynamic process. At birth, there are few tight junctional strands present and a minimal barrier for paracellular diffusion. As the rabbit matures, a more complex network of anastomosing tight junctional strands begins to encircle the cell perimeter under the apical folds. However, even in the mature animal (3 months), there are discontinuities and free ends in the network, thus suggesting that the barrier is not complete even at this stage. Paracellular permeability measurements using 5(6)-carboxyfluorescein as a tracer corroborate these anatomic findings. Endothelial paracellular flux measurements steadily decrease as the rabbit matures from birth to young adult. This indicates that the tight junctional network is increasing in complexity and progressively limiting the flow of substances through the intercellular space.

The developing corneal endothelium: correlation of morphology, hydration and Na/K ATPase pump site density.

The physiology and anatomy of the cornea of the New Zealand white rabbit were studied from birth to young adulthood (3 months). The main objective of the study was to follow the ontogeny of the corneal endothelium and correlate its maturation with the establishment of stromal transparency. With maturity, central corneal thickness increases as do corneal diameter and surface area. Endothelial morphology undergoes marked changes including an increase in cell hexagonality and cell surface area, along with a decrease in cell density and coefficient of variation of cell area. Corneal hydration decreases from a high value at birth to the adult level by 20 days after birth, the time of the onset of stromal transparency. By transmission electron microscopy, corneas of newborn rabbits exhibit an endothelium of irregular cell height with some overlap at the bases of adjacent cells. Apical junctions are incomplete in the neonates. With time the endothelium thins and cells becomes more regular in height, overlap of adjacent cells diminishes, and apical junctions develop. Descemet's membrane is thin in newborns and thickens and becomes more homogenous in appearance with maturation. The abundance of Na/K ATPase pump sites per endothelial cell, as determined by 3H-ouabain binding, increases progressively with age even after the establishment of corneal transparency at 20 days. Scatchard and LIGAND analyses of 3H-ouabain binding data indicate that there is a progressive increase in Bmax with no change in the KD from 7 days to 3 months.