On-Tat Lee

Collagen cross-links reduce corneal permeability.

PURPOSE To investigate the relationship between corneal permeability and nonenzymatic cross-link density. METHODS Corneas were dissected from 90 cadaveric porcine eyes. Samples were incubated for 24 hours with control solution or methylglyoxal at concentrations of 0.01%, 0.10%, and 1.00%. Nonenzymatic cross-link density in treated and control groups was quantified by papain digest and fluorescence spectrophotometry. Control and treated corneas were mounted in a customized Ussing-type chamber connected to vertical tubing, and specific hydraulic conductivity was determined according to the descent of a column of degassed saline at room temperature. Permeability to diffusion of fluorescein in a static chamber was determined for a similar set of corneal samples. RESULTS Methylglyoxal treatment effectively increased nonenzymatic cross-link content, as indicated by the average fluorescence for each group. Specific hydraulic conductivity (m(2)) was reduced with increasing cross-link density. Similarly, the permeability coefficient for the fluorescein solute consistently decreased with increasing methylglyoxal concentration, indicating diffusion impedance resulting from the treatment. CONCLUSIONS Nonenzymatic cross-link density in the cornea can be significantly increased by treatment with methylglyoxal. Porcine cornea showed a nonlinear reduction in solute permeability and specific hydraulic conductivity with increasing cross-link density. This model suggests that age-related nonenzymatic cross-link accumulation can have a substantial impact on corneal permeability.

Ocular Biocompatibility and Structural Integrity of Micro- and Nanostructured Poly(caprolactone) Films

The identification of biomaterials that are well tolerated in the eye is important for the development of new ocular drug delivery devices and implants, and the application of micro- and nanoengineered devices to biomedical treatments is predicated on the long-term preservation within the target organ or tissue of the very small functional design elements. This study assesses the ocular tolerance and durability of micro- and nanostructured biopolymer thin films injected or implanted into the rabbit eye. Structured poly(caprolactone) (PCL) thin films were placed in adult rabbit eyes for survival studies, with serial ophthalmic examinations over 6 months. Morphologic abnormalities and device/tissue reactions were evaluated by histologic studies, and scanning electron microscopy (SEM) of films was used to determine the structural integrity. Structured PCL thin films (20- to 40-μm thick) were constructed to design specifications with 50-μm linear microgrooves or arrays of nanopores with ~30-nm diameters. After up to 9 months of ocular residency, SEM on devices retrieved from the eye showed preservation of micro- and nanostructural features. In ocular safety evaluations carried out over 6 months, serial examinations in 18 implanted eyes showed no evidence of chronic inflammation, cataractogenesis, or retinal toxicity. Postoperative ocular inflammation was seen in 67% of eyes for 1 week, and persistent corneal edema occurred in 1 eye. Histology revealed no ocular inflammation or morphologic abnormalities of ocular tissues. Thin-film/tissue responses such as cellular reaction, fibrosis, or surface biodeposits were not seen. Micro- and nanostructured PCL thin films exhibited acceptable ocular tolerance and maintained the structural integrity of design features while residing in the eye. Thin-film micro- and nanostructured PCL appears to be a feasible biomaterial for intraocular therapeutic applications.

Exogenous collagen cross-linking reduces scleral permeability: modeling the effects of age-related cross-link accumulation.

PURPOSE To investigate the relationship between scleral permeability and nonenzymatic cross-link density. METHODS Scleral discs 18 mm in diameter were dissected from the medial and lateral equatorial regions of 60 cadaveric porcine eyes. Samples were incubated for 24 hours with control solution or methylglyoxal at concentrations of 0.001%, 0.01%, 0.10%, and 1.00%. Nonenzymatic cross-link density in treated and control groups was quantified with the use of papain digest and fluorescence spectrophotometry. Treated scleral discs were mounted in a customized Ussing-type chamber connected to vertical tubing, and specific hydraulic conductivity was determined according to the descent of a column of degassed saline at room temperature. Permeability to diffusion of fluorescein in a static chamber was determined for another set of treated scleral samples. RESULTS Methylglyoxal treatment effectively increased nonenzymatic cross-link content, as indicated by the average fluorescence for each group. Specific hydraulic conductivity (m(2)) was reduced with increasing cross-link density. Similarly, the permeability coefficient for the fluorescein solute consistently decreased with increasing methylglyoxal concentration, indicating diffusion impedance from the treatment. CONCLUSIONS Nonenzymatic cross-link density can be significantly increased by treatment with methylglyoxal. Porcine sclera showed a nonlinear reduction in solute permeability and specific hydraulic conductivity with increasing cross-link density. This model indicates that age-related nonenzymatic cross-link accumulation can have a substantial impact on scleral permeability.

Isolation, Culture, and Characterization of Human Fetal Trabecular Meshwork Cells

Purpose: To isolate and characterize fetal trabecular meshwork (FTM) cells for study in culture. Cultured adult trabecular meshwork (TM) cells often possess a slower rate of growth and restricted number of population doublings, limiting the ability to perform expanded testing. Methods: Fetal eyes from 24-week gestation abortions were delicately dissected to isolate the developing trabecular meshwork tissue. Three primary cultures were achieved and passaged. Light microscopy was used to compare the FTM cells to two cultured adult TM cell lines. Immunocytochemistry and Western blot analysis were utilized to identify specific protein expression. Results: The FTM cells demonstrated similar microscopic characteristics to adult TM cells, including monolayer formation, cobblestone pattern, and comparable size. FTM cells exhibited faster, more consistent doubling times when compared with adult TM cells. They grew rapidly even after passage 8, whereas their adult counterparts slowed significantly with each successive passage and failed to reach confluence at passages 4 to 5. Immunofluorescent staining was positive for actin, vimentin, fibronectin, laminin, aquaporin-1, CD-44, and myocilin in both FTM and adult TM cells. In both fetal and adult cells, Western blots showed substantial increase in myocilin after exposure to dexamethasone. Conclusions: Characterization by microscopy and immunocytochemistry suggest that FTM cells have properties similar to adult TM cells. Fetal tissues may be a useful source of abundant, rapidly dividing FTM cells for in vitro investigation. The ability to do expanded research in this field may contribute to a better understanding of the molecular mechanisms in glaucoma development.

Cross-Linking with Ultraviolet-A and Riboflavin Reduces Corneal Permeability

PURPOSE To investigate the effect of cross-linking treatment on corneal permeability in a live animal model. METHODS Rabbit eyes were selected at random to be left unoperated or to undergo epithelial debridement with or without treatment consisting of cross-linking (CXL) with riboflavin and ultraviolet-A. Nine eyes received a total dose of 3.6 J/cm² and after epithelial healing the corneas were placed in a two-chamber system for quantification of the diffusion of fluorescein compared with controls. Thirty eyes received a total dose of 5.4 J/cm² and, after epithelial healing, in vivo corneal permeability was quantified as the pupillary response over a 30-minute period to a dose of topical pilocarpine compared with controls. RESULTS In the ex vivo assay, the mean permeability coefficient in the CXL group (2.42 × 10⁻⁷) was reduced when compared with the unoperated controls (3.73 × 10⁻⁷; P = 0.007) and to the eyes that received epithelial debridement alone (3.74 × 10⁻⁷; P = 0.01). In the in vivo permeability assay, the change in pupillary diameter at 30 minutes after pilocarpine administration was smaller in the CXL group (-1.9 mm), compared with the epithelial debridement group (-2.6 mm; P < 0.001) and with the unoperated controls (-2.7 mm; P = 0.003). CONCLUSIONS Corneal cross-linking with ultraviolet-A and riboflavin results in a statistically significant reduction in corneal permeability. These findings suggest that dosing of topical medications may need to be increased in eyes with a history of CXL to achieve expected therapeutic effects, and they may have implications for the long-term health of the cornea.

Advanced Glycation End-Product Accumulation Reduces Vitreous Permeability

PURPOSE To evaluate the effect of nonenzymatic cross-linking (glycation) upon the permeability of the vitreous to small- and large-solute diffusion. METHODS Vitreous from freshly excised porcine eyes was treated for 30 minutes with control or 0.01%, 0.1%, or 1% methylglyoxal (MG) solution. The efficacy of the glycation regimen was verified by measuring nonenzymatic cross-link density by fluorescence in the vitreous samples. Resistance to collagenase digestion as well as N(ε)-(carboxyethyl) lysine (CEL) content were also measured. The permeability coefficient for fluorescein and fluorescein isothiocyanate (FITC)-IgG diffusion through 3 mL of the vitreous samples was determined by using a custom permeability tester. RESULTS Vitreous cross-linking with MG treatment was confirmed by increased fluorescence, increased CEL concentration, and increased resistance to collagenase digestion. Vitreous glycation resulted in a statistically significant decrease in the permeability coefficient for fluorescein diffusion when either 0.1% or 1% MG solution was used (5.36 ± 5.24 × 10(-5) cm s(-1), P = 0.04; and 4.03 ± 2.1 × 10(-5) cm s(-1), P = 0.001; respectively, compared with control, 9.77 ± 5.45 × 10(-5) cm s(-1)). The permeability coefficient for diffusion of FITC-IgG between control (9.9 ± 6.37 × 10(-5) cm s(-1)) and treatment groups was statistically significant at all MG concentrations (0.01% MG: 3.95 ± 3.44 × 10(-5) cm s(-1), P = 0.003; 0.1% MG: 4.27 ± 1.32 × 10(-5) cm s(-1), P = 0.004; and 0.1% MG: 3.72 ± 2.49 × 10(-5) cm s(-1), P = 0.001). CONCLUSIONS Advanced glycation end-product (AGE) accumulation reduces vitreous permeability when glycation is performed in ex vivo porcine vitreous. The permeability change was more pronounced for the larger solute, suggesting a lower threshold for AGE-induced permeability changes to impact the movement of proteins through the vitreous when compared with smaller molecules.

Ultrastructure and fluid flow physiology of fetal trabecular meshwork cells.

Purpose: To assess the ultrastructural and fluid flow characteristics of cultured trabecular meshwork (TM) cells derived from fetal sources. Methods: Fetal eyes were carefully dissected to isolate the developing TM tissue for culture. Immunostaining was used to assess the expression of the junction-associated proteins zonula occludens-1 (ZO-1) and occludin. Fetal and adult TM cells were grown to confluence on permeable membranes for both flow and ultrastructural studies. Fluid flow resistance was measured by permeation of horseradish peroxidase (hrp) activity and hydraulic conductivity (HC) experiments. The effects of dexamethasone (Dex) on permeability and HC were also evaluated. Results: ZO-1 and occludin are expressed in the TM region of tissue sections and at cell borders in cultured fetal and adult TM cells. Transmission electron microscopy demonstrated that cultured TM cells possessed numerous mitochondria, electron-dense bodies, surface microvilli, and adherens and gap junctions. The permeation of hrp across fetal TM cell monolayers (0.030 ± 0.010) and of adult TM cells (0.031 ± 0.010) had similar values for absorbance at 470 nm (p = 0.83, 95% CI: −0.004, 0.005). Dex treatment significantly reduced the permeability to 0.022 ± 0.008 (p = 0.002) and 0.018 ± 0.009 (p = 0.004) for fetal and adult TM cells, respectively. The average HC (μ l/min/mmHg/cm2) of fetal cells (2.78 ± 1.03) and of the adult cells (2.15 ± 1.31) was not significantly different (p = 0.24, 95% CI: −1.01, 0.26). Dex treatment significantly reduced HC in both fetal (1.24 ± 0.72, p = 0.0004) and adult (1.29 ± 0.29, p = 0.00001) TM. Conclusions: Cultured fetal TM cells exhibited similar expression of junctional proteins and ultrastructural features as their adult counterparts. The permeability and HC of the fetal cells were similar to their older adult counterparts. Dex treatment induced increased fluid flow resistance in both cell types. These cells may serve as a source for in vitro studies of meshwork physiology.