Dayle H. Geroski

Human Sclera: Thickness and Surface Area

PURPOSE To assess the mean thickness and surface area of human sclera. METHODS Fifty-five formalin-fixed eye bank eyes were hemisected from anterior to posterior. Cross-sectional slides were taken to include a millimeter scale ruler in each photograph. Slide photographs were projected and the scleral silhouette sketched. Mean scleral thickness measurements with standard deviation were obtained. Twenty-five human eye bank eyes were used to determine total scleral surface area by either a computerized tracing method (17 globes) or volumetric calculations (eight globes) using fluid displacement. RESULTS Mean scleral thickness +/- SD was 0.53 +/- 0.14 mm at the corneoscleral limbus, significantly decreasing to 0.39 +/- 0.17 mm near the equator, and increasing to 0.9 to 1.0 mm near the optic nerve. The mean total scleral surface area by surface area computerized tracings was 16.3 +/- 1.8 cm2 and, by the volume displacement method, was 17.0 +/- 1.5 cm2. CONCLUSIONS Scleral thickness and surface area measurements from cadaver eyes are important for ophthalmic surgeons and have implications for transscleral diffusion.

Transscleral drug delivery for posterior segment disease.

Exciting new treatments are being developed for retinal degenerations and posterior segment eye disease. The successful treatment of these visually devastating diseases will likely require delivering effective doses of pharmacologic agents to the posterior segment, possibly in conjunction with surgical or genetic interventions. Currently, the treatment of diseases affecting the posterior segment is limited by the difficulty in delivering effective doses of drugs to target tissues in the posterior vitreous, retina or choroid. This review summarizes recent laboratory and clinical studies that indicate that transscleral delivery of therapeutic solutes might be an effective means of achieving therapeutic concentrations of these agents in the posterior eye.

Drug delivery through the sclera: effects of thickness, hydration, and sustained release systems.

The purpose of this study was to determine whether trans-scleral pressure affects scleral solute permeability by altering scleral thickness or hydration, and to investigate the sustained release delivery of dexamethasone. Scleral sections from donor human globes were mounted for in vitro flux studies. Scleral thickness and hydration were measured as functions of trans-scleral pressure. For the sustained release studies, 3H-dexamethasone in pluronic F-127 gel or in fibrin sealant was added to the episcleral side of the tissue and flux studies were performed. While scleral thickness showed a tendency to decrease with increasing pressure, a significant decrease in thickness was measured only at a trans-scleral pressure of 60 mmHg. No significant changes in scleral hydration were measured over the range of trans-scleral pressures studied. The apparent permeability constants (Ktrans) of human sclera for 3H-dexamethasone in BSS plus, fibrin sealant and F-127 gel were 11.5 x 10(-6), 7.3 x 10(-6), and 1.5 x 10(-6) cm sec(-1), respectively. Human scleral permeability to dexamethasone differed significantly among the three vehicles (p < 0.0001). Cumulative delivery of dexamethasone from BSS plus, F-127 gel, and fibrin sealant were 85.0, 29.3, and 67.9% at 20 hr, respectively. Scleral hydration was unaffected by trans-scleral pressures. Scleral thinning was only observed at 60 mmHg. Trans-scleral pressures below 60 mmHg would not be expected to significantly affect the permeability of the tissue to solutes in the size range of conventional drugs. F-127 gel and fibrin sealant provided a slow, relatively uniform sustained release through a 24 hr period. These systems might be employed to achieve sustained therapeutic levels of drugs to the posterior segment of eye.

In Vitro Human Scleral Permeability of Fluorescein, Dexamethasone-Fluorescein, Methotrexate-Fluorescein and Rhodamine 6G and the Use of a Coated Coil as a New Drug Delivery System

PURPOSE To determine the in vitro human scleral permeability of several dyes and drugdye combinations with varying molecular weights (MW) and lipid solubilities (fluorescein, dexamethasone-fluorescein, methotrexate-fluorescein, and rhodamine). Coils coated with rhodamine were also evaluated for scleral permeability and sustained release. METHODS Scleral sections excised from moist chamber stored human globes were mounted in a 2-compartment perfusion chamber. A small depot of drug/dye (100 microl of 10(-4) M fluorescein, dexamethasone-fluorescein, methotrexate-fluorescein or rhodamine) or a coated coil in 100 microl of BSS was added to the episcleral surface while perfusing BSS to the choroidal side. The perfusate was collected and measured for fluorescence. Permeability was calculated as Ktrans from the flux measurements. RESULTS Ktrans values (cm/sec, mean +/- SE) for the studied dyes and drug-dye combinations were 5.21 +/- 0.71 x 10(-6) for fluorescein, 1.64 +/- 0.17 x 10(-6) for dexamethasone-fluorescein, 3.36 +/- 0.62 x 10(-6) for methotrexate-fluorescein, 1.86 +/- 0.39 x 10(-6) for rhodamine and 2.18 +/- 0.23 x 10(-6) for the rhodamine from the coils. We found a significant difference between the permeability of the sclera to fluorescein and dexamethasone-fluorescein (P < 0.001), methotrexate-fluorescein (P < 0.05) and rhodamine (P < 0.001). Steady state flux was observed from the rhodamine coil. CONCLUSION The rank order of scleral permeability to the studied dyes is as follows: fluorescein > methotrexate-fluorescein > rhodamine coil > rhodamine 6G > dexamethasone-fluorescein. Differences in scleral permeability are related to MW and lipid solubility. Prolonged transscleral diffusion of rhodamine delivered by solution and by coil are similar.

Transscleral permeability and intraocular concentrations of cisplatin from a collagen matrix.

Abstract This study determined the in vitro permeability of cisplatin through isolated human sclera as delivered by a collagen matrix vehicle. Short-term and long-term intraocular levels of cisplatin were also measured in the rabbit eye after a subconjunctival injection. Cisplatin in either a collagen matrix vehicle or a control balanced salt solution (BSS) vehicle was applied to human sclera mounted in a specially designed in vitro perfusion chamber. The amount of cisplatin that diffused across the sclera was measured in hourly samples for 24 h using atomic absorption spectrometry. In vivo studies were also performed in Dutch Belted rabbits given subconjunctival injections of cisplatin in collagen matrix or in BSS. Eyes were enucleated at 1.5 h and 2 weeks after injection, frozen, and dissected to determine the intraocular cisplatin concentrations. Cisplatin had a peak in vitro scleral permeability constant of 8.3±1.2×10−6 and 20.1±1.8×10−6 cm/s, delivered in collagen matrix and in BSS, respectively (mean±S.D.). At the end of the in vitro experiments, 35.9±4.6% of the cisplatin remained in the collagen matrix, while 0.8±0.2% remained in the BSS vehicle. Subconjunctival injection of cisplatin in the collagen matrix vehicle achieved 3.3±0.1 μg/ml in the vitreous humor at 1.5 h and 0.1±0.1 μg/ml at 2 weeks. This vehicle also achieved a cisplatin concentration of 73.5±23.9 μg/mg in the choroid and retina at 1.5 h and 3.2±1.3 μg/mg at 2 weeks. Compared to BSS, the collagen matrix vehicle provided a more controlled release of cisplatin, and after subconjunctival injection into rabbits, attained higher drug levels in several ocular tissues.

The corneal epithelium after optisol-GS storage.

The objective of this study was to evaluate the epithelium of human corneas stored in Optisol-GS (Chiron Intraoptics, Irvine, CA) for extended periods (2-34 days). Human corneas stored in Optisol-GS (n = 64) were obtained from the Georgia Eye Bank. Corneal epithelial viability was assessed by using the Calcein-AM (Molecular Probes, Inc., Eugene, OR) ethidium homodimer stain, a fluorescent assay used to distinguish live from dead cells. Scanning and transmission electron microscopy was used to evaluate epithelial ultrastructure. The results showed that corneas stored up to 6 days in Optisol-GS had minimal damage of the epithelium. Calcein-AM ethidium homodimer staining showed 20-25% epithelial damage. Corneas stored 7-10 days had a further increase in epithelial damage (30-35%). Corneas stored for 11-15 days had marked increases in epithelial damage (40-50%), and corneas stored 16-34 days showed significant epithelial damage (60-70%). The data show that corneas stored in Optisol-GS are able to maintain the epithelium up to 6 days. A gradual decrease in epithelial viability and loss of epithelial cells occurs in corneas stored 6-10 days. Corneas stored for > 10 days have a marked loss of epithelial cells with extensive epithelial damage.

Effects of laser in situ keratomileusis (LASIK) on the corneal endothelium

PURPOSE To assess the effects of laser in situ keratomileusis (LASIK) on the corneal endothelium. METHODS In a prospective study, the corneal endothelium of 98 eyes of 65 consecutive patients (mean age, 41 years; range, 22 to 66 years) was photographed before, 2 weeks after, and 12 weeks after LASIK for the correction of 2.75 to 14.5 diopters of myopia. Theoretical ablation depths were 200 to 330 microm below the corneal surface. Cell density, coefficient of variation, and percent of hexagonal cells were determined using 150 to 200 cells from each image. Eighty-eight eyes (91%) of 59 patients had a history of contact lens wear. RESULTS The mean+/-SD preoperative endothelial cell density was 2,549+/-365 cells per mm2, and the mean coefficient of variation was 0.35+/-0.06. There was no statistically significant change in the mean endothelial cell density or mean coefficient of variation of cell size at the 2-week (2,561+/-360 cells per mm2 and 0.35+/-0.06) or 12-week (2,541+/-364 cells per mm2 and 0.35+/-0.05) postoperative examinations. The percent of hexagonal cells was not significantly changed 2 weeks postoperatively; however, 12 weeks postoperatively (P=.0413, two-tailed t test), the percent of hexagonal cells was decreased by 1%. CONCLUSIONS Corneal endothelial cell density and morphology were unchanged 2 and 12 weeks after LASIK for the correction of up to 14.5 diopters of myopia. In this LASIK study, the correction of up to 14.5 diopters of myopia appears to cause no clinically significant effect on corneal endothelial cell density or morphology.

Corneal Endothelial Cytoskeletal Changes in F-Actin With Aging, Diabetes, and After Cytochalasin Exposure

We investigated the changes in endothelial cytoskeletal F-actin that occur with aging, diabetes, and exposure to cytochalasin D. Rabbit corneas, human donor corneas (with or without polymegethism), and corneas of diabetic individuals were studied. Endothelial F-actin was stained using nitrobenzoxadiazole-phallacidin. Results of these experiments demonstrated that F-actin of the rabbit and human corneal endothelium was arranged in linear circumferential strands that formed a hexagonal array. After in vitro perfusion of cytochalasin D to the corneal endothelium, the F-actin became randomly distributed throughout the cytoplasm, the hexagonal shape of the endothelial cell was disrupted, and endothelial permeability to carboxyfluorescein increased. Changes in F-actin were also observed in the endothelium of the human corneas with polymegethism, and in donor tissue having had previous posterior chamber intraocular lens implantation. The corneas of diabetic individuals also showed marked irregular F-actin fibers crossing the endothelial cell cytoplasm. These abnormal patterns of F-actin may contribute in part to the polymegethism observed in the corneal endothelial cells and may be the result of constant stress in cell volume regulation, particularly in the corneas of diabetic individuals.

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. Descemets 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.

Effect of inflammation on the corneal endothelial pump and barrier

Corneal thickness is a reflection of endothelial barrier and pump functions. The corneal edema that occurs during intraocular inflammation is a consequence of the breakdown of one or both of these parameters. Results of this study demonstrate that, during intraocular inflammation induced by an intravitreal injection of bovine serum albumin (BSA), the permeability of rabbit corneal endothelia to inulin was increased. By comparison, treatment with oral aspirin and/or subconjunctival triamcinolone acetonide prevented the endothelial barrier breakdown induced by the BSA. Concomitant with the loss of the barrier function, endothelial ouabain binding decreased in the BSA injected eye, indicating a reduction in endothelial Na/K ATPase pump site density. A subconjunctival injection of triamcinolone prevented this decrease in pump sites. The increase in endothelial permeability and the decrease in pump site density correlated with an increase in corneal thickness. It can be concluded that the intraocular inflammation induced by BSA effects corneal edema by both an increase in endothelial permeability and a decrease in Na/K ATPase pump site density. Subconjunctival triamcinolone is effective in preventing this response.