Evaluation of endothelial mucin layer thickness after phacoemulsification with next generation ophthalmic irrigating solution.

PURPOSE: To evaluate human corneal endothelial mucin layer thickness and ultrastructure after phacoemulsification and irrigation-aspiration with either next generation ophthalmic irrigating solution (NGOIS) or BSS PLUS. METHODS: Paired human corneas were mounted in an artificial anterior chamber, exposed to 3 minutes of continuous ultrasound (US) at 80% power using the Alcon SERIES 20000 LEGACY surgical system (n = 9) or to 2 minutes of pulsed US at 50% power, 50% of the time at 20 pps using the Alcon INFINITI Vision System (n = 5), and irrigated with 250 mL of either NGOIS or BSS PLUS. A control group of paired corneas did not undergo phacoemulsification or irrigation-aspiration (n = 5). Corneas were divided and fixed for mucin staining or transmission electron microscopy. Mucin layer thickness was measured on the transmission electron microscopy prints. RESULTS: The mucin layer thickness in the continuous phaco group was 0.77 +/- 0.02 microm (mean +/- SE) with NGOIS and 0.51 +/- 0.01 microm with BSS PLUS (t test, P < 0.001). The mucin layer thickness in the pulsed phaco group was 0.79 +/- 0.02 microm with NGOIS and 0.54 +/- 0.01 microm with BSS PLUS (P < 0.001). The mucin layer thickness in the untreated control group was 0.72 +/- 0.02 microm. The endothelial ultrastructure was normal in all corneas. CONCLUSIONS: In this in vitro corneal model, NGOIS, due to its lower surface tension and higher viscosity, preserved endothelial mucin layer thickness better than BSS PLUS with both the INFINITI Vision System (pulsed US) and the LEGACY surgical system (continuous US).

Effect of mitomycin C on the corneal endothelium when used for corneal subepithelial haze prophylaxis following photorefractive keratectomy.

PURPOSE: To evaluate the potential effect of topical mitomycin C (MMC) on the corneal endothelium of myopic patients undergoing photorefractive keratectomy (PRK). METHODS: Sixteen eyes with a planned ablation depth >75 microm underwent PRK followed by 0.02% MMC applied for 12 seconds using a methylcellulose sponge. Endothelial specular microscopy was performed with the Keeler-Konan specular photomicroscope in 16 eyes before and at least 1 year after surgery. Mean follow-up was 18 months (range: 12 to 24 months). Mean cell density, coefficient of variation of mean cell area, and percentage of hexagonal cells were measured and calculated using computerized morphometric analysis. RESULTS: Mean endothelial cell densities before and after surgery were 2882 +/- 783 cells/mm2 (range: 1511 to 4022 cells/mm2) and 2867 +/- 588 cells/mm2 (range: 1638 to 3881 cells/mm2), respectively (P > .05). Mean coefficient of variation before and after surgery was 0.30 +/- 0.07 (range: 0.23 to 0.49) and 0.26 +/- 0.04 (range: 0.22 to 0.33), respectively (P=.06). Mean percentage of hexagonal cells before and after surgery was 61% +/- 6.8% (range: 47% to 70%) and 66% +/- 6.7% (range: 54% to 75%), respectively. CONCLUSIONS: Administration of MMC for haze prophylaxis following PRK did not have a significant effect on quantitative endothelial cell density or qualitative morphometric parameters in this study.

Interface fluid syndrome in human eye bank corneas after LASIK: causes and pathogenesis.

PURPOSE: To evaluate the effects of corneal edema on human donor corneas that had previous LASIK using a laboratory model with histologic and ultrastructural correlations. DESIGN: Experimental study. PARTICIPANTS: Thirty human eye bank corneas from 15 donors (mean age +/- standard deviation, 49.9+/-8.9 years) who had had previous LASIK surgery (2-8 years before death). METHODS: The corneas were mounted in an artificial anterior chamber and the corneal endothelium was perfused for up to 5.0 hours with 0.9% saline solution (endothelial cell damage group) or BSS Plus at a pressure of 15 mmHg (control group), or BSS Plus at a pressure of 55 mmHg (high-pressure group). The corneas were evaluated by confocal and specular microscopy before, during, and at the end of the experimental period. Subsequently, the specimens were evaluated by light and electron microscopy. MAIN OUTCOME MEASURES: Corneal thickness, reflectivity, histology, and ultrastructure. RESULTS: Endothelial cell damage resulted in an increased (141.5+/-38.8 microm) total corneal thickness relative to controls (52.3+/-33.7 microm), whereas high pressure resulted in a decreased thickness (24.8+/-14.1 microm) relative to controls. This ultimately was due to swelling of the LASIK interface in both groups and swelling of the residual stromal bed (RSB) in the endothelial cell damage group or compression of the RSB and, possibly, the flap in the high-pressure group. A significant increase in corneal reflectivity at the LASIK interface occurred in both groups, primarily due to varying degrees of fluid accumulation and associated hydropic keratocyte degeneration, as well as increased corneal reflectivity in the RSB only in the endothelial cell damage group. CONCLUSIONS: After LASIK surgery, edematous corneas preferentially hydrate and swell in the paracentral and central interface wound, commonly resulting in a hazy corneal appearance primarily due to keratocyte hydropic degeneration. More severe corneal edema is characterized by the formation of an optically empty space corresponding to an interface fluid pocket. The spectrum of interface fluid syndrome can be described in 3 stages.

Antibody blockade of junctional adhesion molecule-A in rabbit corneal endothelial tight junctions produces corneal swelling.

PURPOSE: The ultrastructure of tight junctions in the corneal endothelium has been studied extensively, yet little is known about their molecular composition. Junctional adhesion molecule-A (JAM-A) is a tight junction-associated adhesion protein previously implicated in tight junction assembly and regulation of barrier function. In this study, we sought to investigate the expression and function of JAM-A in the corneal endothelium. METHODS: Immunofluorescence confocal microscopy was used to investigate expression of JAM-A and the related proteins JAM-C, CAR, and AF-6 in the rabbit corneal endothelium. Corneal endothelial perfusion specular microscopy was then used to test the effects of antibodies to JAM-A on corneal swelling. RESULTS: The expression of JAM-A was observed in the tight junctions of rabbit corneal endothelium in a localization pattern identical with that of ZO-1, a known marker of the tight junction and binding partner of JAM-A. Expression of related proteins JAM-C and CAR (Coxsackie and adenovirus receptor) was also observed in the corneal endothelium, but their distribution was diffuse and not limited to the tight junction. Expression of AF-6, a known binding partner of JAM-A, was also observed in the tight junction in a pattern similar to ZO-1. Last, functional experiments were performed in which a monoclonal antibody to JAM-A was shown to increase rabbit corneal swelling by 63% compared with the control. CONCLUSIONS: The results provide new evidence that JAM-A and its binding partner AF-6 are expressed in tight junctions of the corneal endothelium and that JAM-A has a major role in maintaining the corneal endothelial barrier function.

Ex vivo confocal microscopy of human LASIK corneas with histologic and ultrastructural correlation.

OBJECTIVE: To perform confocal microscopy on postmortem human LASIK corneas and correlate these findings to histologic and ultrastructure evaluations. DESIGN: Prospective, consecutive, observational case series. PARTICIPANTS: Ninety postmortem LASIK corneas (47 patients) were evaluated for histopathology, of which 22 consecutive corneas (12 patients) were also evaluated by confocal microscopy. Six normal corneas (3 patients) served as controls. METHODS: This observational case series involving 22 corneas from 12 patients with postoperative intervals from 1 month to 6.5 years after LASIK surgery were collected. The corneas were mounted in an artificial anterior chamber and perfused with balanced salt solution before confocal microscopy was performed on the center of the cornea. The corneas were then bisected and processed for light and transmission electron microscopy. RESULTS: Confocal microscopy, along with histologic and ultrastructural correlations, demonstrated that the most prevalent alterations in the centers of LASIK corneas were a slightly thickened epithelium caused by focal basal epithelial cell hypertrophic modifications, random undulations in Bowman's layer over the flap surface, and a variably thick hypocellular primitive stromal interface scar. By using confocal microscopy, the interface wound was easily identified in 100% of the cases because numerous brightly reflective interface particles were always present in the hypocellular primitive stromal scar. These particles were found primarily to consist of organic cellular constituents, some of which were transient in nature. CONCLUSION: After LASIK, active stromal wound healing in the central cornea results in the production of a hypocellular primitive stromal scar, whereas secondary tissue adjustments seem to cause the Bowman's layer undulations and the subsequent epithelial cell modifications. Most of the interface particles revealed by confocal microscopy in the region of the stromal scar are organic in nature and presumably innocuous to the cornea.

Increased endothelial cell density in the paracentral and peripheral regions of the human cornea.

PURPOSE: To systematically investigate the central, paracentral, and peripheral endothelial cell density (ECD) in normal human corneas. DESIGN: Observational case series and experimental study. METHODS: Noncontact specular microscopy was undertaken to determine the ECD of the central, paracentral (2.7 +/- 0.2 mm from center) and peripheral (4.7 +/- 0.2 mm from center) regions of the cornea of 48 normal eyes. The ECDs of central and peripheral regions were also determined with contact specular microscopy in 21 normal eyes and a group of 30 Optisol-GS eye bank corneas were evaluated with alizarin red stain. Histologic ECD of 13 Optisol-GS stored corneas were also determined. RESULTS: Paracentral and peripheral ECD measured with the noncontact specular microscope were 5.8% (P <.01) and 9.6% (P <.001) increased compared with central ECD. Superior peripheral ECD was increased compared with the other three peripheral quadrants (P <.05) and was 15.9% higher than central ECD. Contact specular microscopy showed an increase of 8.9% in the peripheral ECD from the center. Alizarin red stained corneas confirmed the specular microscopy numbers with a 9.2% increase in the paracentral region, and a 17.2% increase in the peripheral region. Histological cross sections of human corneas also showed a 22.9% increase in peripheral ECD compared with the central region. CONCLUSIONS: The human cornea has an increased ECD in the paracentral and peripheral regions of cornea compared with the central region. The superior peripheral region of the corneal endothelium has the largest increase in ECD. These data on normal endothelial cell distribution in the human cornea are especially significant as they relate to new surgical techniques and endothelial wound repair.

Effect of indocyanine green intraocular stain on human and rabbit corneal endothelial structure and viability. An in vitro study.

PURPOSE: To evaluate the direct effect of intraocular indocyanine green (ICG) on endothelial cell function, ultrastructure, and viability in human and rabbit corneas. SETTING: A laboratory evaluation study. METHODS: Paired human and rabbit corneas were mounted in an in vitro specular microscope for endothelial cell perfusion. One corneal endothelium was perfused with 25 mg ICG dissolved in 0.5 mL aqueous solvent in 4.5 mL balanced salt solution (BSS(R)) for 3 minutes followed by washout with a control solution. The percentage of ICG exposed to the corneal endothelium was 0.5%. The paired cornea was perfused with the same solution without ICG, followed by the washout. The corneas were fixed for scanning and transmission electron microscopy (TEM). In another group, the endothelial viability was determined using a live cell/dead cell assay. RESULTS: In rabbit corneas, the mean corneal swelling rate was 12.9 microm/h +/- 1.2 (SEM) in the ICG corneas and 2.8 +/- 1.9 microm/h in the controls. Scanning electron microscopy and TEM revealed a normal endothelial cell mosaic. The control electron micrographs were similar. In human corneas, the mean swelling rate was 19.1 +/- 2.8 microm/h in the ICG corneas and 19.2 +/- 2.6 microm/h in the controls. Scanning electron microscopy and TEM revealed intact junctions with slight cellular vacuolization, similar to that in the controls. In the live cell/dead cell subgroup, the mean damage was 17.3% +/- 1.7% in the ICG-exposed corneas and 22.0% +/- 8.9% in the controls. CONCLUSIONS: Three-minute exposure to ICG in BSS had no adverse effect on corneal endothelial function, ultrastructure, or viability in human and rabbit corneas. This study provides a safety profile for the corneal endothelium when ICG is used as an intraocular tissue stain in ophthalmic surgery.