Tracy T. Nguyen

Overnight orthokeratology: visual and corneal changes.

Purpose. To achieve an optimal fit with reverse geometry Contex OK lenses and to determine a time course for and the stability of visual and corneal changes in achieving maximal refractive, corneal curvature, and corneal thickness changes after overnight wear of OK B and D series lenses. Methods. This investigation was conducted under an Food and Drug Administration IDE G000059. Both eyes of 10 subjects were fitted with the lenses, and uncorrected visual acuity, refractive correction, contrast sensitivity, corneal curvature, and corneal thickness were measured at baseline and at 1 day, 1week, 1 month, and 3 months after lenses were worn. Except for baseline, data were collected at four different times during the day, immediately following lens removal and 4, 8, and 12 hours after lens removal. Results. The results from eight subjects showed that uncorrected visual acuity, refractive correction, contrast sensitivity, and corneal curvature all changed significantly (P =0.01) overnight. By the end of 1 week, all corneal and visual changes had reached a maximal level and remained fairly stable during the day. These changes were sustained at 3 months. The epithelial thickness data from four subjects showed that the corneal epithelial thickness was reduced by approximately 19 &mgr;m after 3 months of lens wear. Conclusions. Successful fitting of OK B and D series lenses requires a thorough understanding of the lens–cornea relationship. Full effect of overnight orthokeratology is achieved by the end of 1 week. The visual and corneal changes remain stable for all waking hours of the day and allow patients to enjoy excellent device-free vision (20/20).

Overnight orthokeratology: refractive and corneal recovery after discontinuation of reverse-geometry lenses.

Purpose. To determine the refractive and corneal topographic recovery after the use of reverse-geometry contact lenses for overnight orthokeratology. Methods. Both eyes of 15 subjects were fitted with reverse-geometry contact lenses that were worn by the subjects for 1 month. Uncorrected visual acuity, refractive correction (sphere and spherical equivalent), corneal curvature, and corneal thickness were measured during this time and for 2 weeks after discontinuation of lens wear. Results. Ten subjects completed the investigation. Uncorrected visual acuity, refractive correction, and corneal curvature had changed significantly (P= 0.01) after 1 month of lens wear. By the end of 1 month, central corneal thickness was significantly thinner than the baseline value (P= 0.01), but it recovered fully after one night of no lens wear. Recovery of corneal curvature was complete 1 week after lens wear was discontinued. Refractive correction and binocular uncorrected visual acuity recovered fully after 2 weeks. Monocular uncorrected visual acuity remained significantly (P= 0.01) different from baseline acuity 2 weeks after lens discontinuation. Conclusions. Full effect of overnight orthokeratology in low myopes is achieved within 1 week of initiating use of reverse-geometry lenses. Recovery after short-term use of reverse-geometry lenses is rapid for corneal thickness and corneal curvature. Refractive correction and binocular uncorrected visual acuity recovered fully after 2 weeks. Monocular uncorrected visual acuity was the slowest to recover and had not achieved full recovery after 2 weeks.

Telomerase Immortalization of Human Corneal Endothelial Cells Yields Functional Hexagonal Monolayers

Human corneal endothelial cells (HCEnCs) form a monolayer of hexagonal cells whose main function is to maintain corneal clarity by regulating corneal hydration. HCEnCs are derived from neural crest and are arrested in the post-mitotic state. Thus cell loss due to aging or corneal endothelial disorders leads to corneal edema and blindness–the leading indication for corneal transplantation. Here we show the existence of morphologically distinct subpopulations of HCEnCs that are interspersed among primary cells and exhibit enhanced self-renewal competence and lack of phenotypic signs of cellular senescence. Colonies of these uniform and hexagonal HCEnCs (HCEnC-21) were selectively isolated and demonstrated high proliferative potential that was dependent on endogenous upregulation of telomerase and cyclin D/CDK4. Further transduction of HCEnC-21 with telomerase yielded a highly proliferative corneal endothelial cell line (HCEnT-21T) that was devoid of oncogenic transformation and retained critical corneal endothelial cell characteristics and functionality. This study will significantly impact the fields of corneal cell biology and regenerative medicine.

Variability in hypoxia-induced corneal swelling is associated with variability in corneal metabolism and endothelial function.

Purpose. To determine if the variability in contact lens-induced corneal swelling is associated with variability in corneal oxygen consumption (QC) or corneal endothelial function. Methods. Corneal swelling was induced in 30 non-contact lens wearers by 2 hours of closed-eye contact lens wear, using thick (oxygen transmission [Dk/t] = 4.0 × 10−9) and thin (Dk/t = 12 × 10−9) hydrogel lenses of identical design. Following the induction of swelling, corneal thickness was continually measured by pachymetry until open-eye steady-state (OESS) thickness was achieved. The percentage of recovery per hour was calculated as a measure of endothelial function. Tear oxygen tension (PO2) beneath the hydrogels was measured in the open eye and after 5 minutes of eye closure to obtain estimates of QC. A change in corneal pH during eye closure while wearing the hydrogels was used as a measure of hypoxic acidosis. Associations between corneal swelling and endothelial cell density or corneal epithelial thickness were also tested. Results. There were modest but significant (P < 0.05) correlations between thick-lens corneal swelling and thick-lens closed-eye PO2 (r = −0.36); thin lens corneal swelling and thin-lens closed-eye and open-eye PO2 (r = −0.40 and −0.39, respectively). Corneal swelling also increases with increasing QC (r = + 0.29 to + 0.33). Corneal swelling was associated with the decrease in pH during lens wear (r = + 0.30 and + 0.20 for thick and thin lenses, respectively). Thick- and thin-lens corneal swelling was significantly associated with percentage of recovery per hour (r = −0.40 and −0.34, respectively). Multiple regression analysis of corneal swelling with PO2 and percentage of recovery per hour suggested an additive effect, however the effects were not significant. There was a modest inverse association between corneal swelling and OESS, however there were no associations between corneal swelling and endothelial cell density or epithelial thickness. Conclusions. The variability in contact lens-induced corneal swelling is associated with both corneal metabolic activity (QC and pH) and endothelial function (percentage of recovery per hour). Our interpretation is that individuals with larger QC produce more lactic acid (i.e., more swelling) whereas stronger endothelial function resists swelling. The modest correlations, however, suggest that other factors also are involved in explaining the phenomenon of corneal swelling.

Hypoxia Protects Human Corneal Endothelium from Tertiary Butyl Hydroperoxide and Paraquat-Induced Cell Death In Vitro

To develop new and safe approaches to protect and preserve the function of the human corneal endothelium (HCE), we have established an in vitro model of cell loss using an HCE cell line and have examined the potential for hypoxia conditioning to protect HCE from death induced by two ROS generating cytotoxins (tertiary butyl hydroperoxide [tBHP] and paraquat [PQ]). Cell death was assessed by flow cytometric analysis of Annexin-V (An) and propidium iodide (PI) double staining and oligonucleosome production. Mitochondrial membrane potential (MMP) was measured with JC-1 fluorescent dye to determine possible associations between MMP preservation and cell survival. PQ and tBHP both induced HCE cell death in a dose-dependent manner, with 48% and 32% of An+ cells observed with 60 μM tBHP and 500 μM PQ, respectively. In addition, this level of tBHP and PQ resulted in 66% and 49% decreases in MMP, respectively. Hypoxia (0.6% ± 0.1% oxygen) pretreatment (8 hrs) significantly reduced An+ cells caused by 60 μM tBHP to 15%, whereas hypoxia had no effect on the decreased MMP. Hypoxia pretreatments (24 hrs) slightly reduced An+ cells caused by 500 μM PQ to 25% and completely prevented the induced MMP reduction. Conversely, hypoxia posttreatment (24 hrs) resulted in a greater inhibition of cell death than pretreatment and yet failed to prevent the PQ-induced MMP collapse. In conclusion, these results suggest that hypoxia can sufficiently protect HCE cells against cell death caused by tBHP and PQ, although no direct link between hypoxia cell protection and MMP preservation was observed.

Knockdown of NBCe1 In Vivo Compromises the Corneal Endothelial Pump

PURPOSE To evaluate the role of the sodium bicarbonate cotransporter (NBCe1) as a component of the corneal endothelial pump in the in vivo rabbit eye. METHODS Lentiviruses with NBCe1 shRNA and GFP expression cassettes were injected intracamerally. Knockdown efficacy was determined 1 week to 4 weeks later by immunofluorescence, Western blot analysis, and PCR. Functional effects were monitored by corneal thickness (CT) and brinzolamide sensitivity. RESULTS Within 24 hours there was a modest anterior chamber inflammation that resolved within 48 hours. At 4 × 10(6) IFU, more than 95% of the corneal endothelial surface showed GFP fluorescence above background within 7 days. At 14 to 21 days, signs of anterior chamber inflammation reemerged, and endothelial cell GFP fluorescence disappeared within 40 days after injection. The second phase of inflammation could be avoided by using GFP-less viruses. There was no significant difference in CT between scrambled sequence and NBCe1 shRNA-injected eyes over 3 weeks. Two drops of 1% brinzolamide produced 7.85% ± 3.3% corneal swelling within 5 hours of topical instillation. However, in corneas showing more than 25% NBCe1 knockdown (30 of 42 rabbits; 59% ± 15% knockdown), corneal swelling was significantly higher (10.1% ± 2.9%) relative to control eyes. CONCLUSIONS FIV-based lentiviral vectors can transfect CE with shRNA in rabbits. The response to GFP is consistent, with previous studies showing the production of anti-GFP antibodies. Partial knockdown of NBCe1 did not affect baseline CT, which is consistent with the corneal endothelium having a substantial functional reserve. Provocative testing using, brinzolamide, however, revealed an underlying deficiency, confirming the importance of NBCe1 bicarbonate transport and demonstrating the concerted action between NBCe1 and carbonic anhydrases.