Corneal cell response after flap creation using a mechanical microkeratome or a 200 kHz femtosecond laser.

PURPOSE: To compare the inflammatory cell response within the corneal flap interface created by a mechanical microkeratome and a femtosecond laser. SETTING: Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany. DESIGN: Experimental in vitro study. METHODS: Corneoscleral buttons of 12 enucleated human eyes not suitable for transplantation were put into organ culture. Corneal flaps were created using a 200 kHz femtosecond laser (Visumax) (femtosecond group) or a mechanical microkeratome (Amadeus) (microkeratome group). Flaps were not lifted after treatment. In 2 corneas, no treatment was performed (control group). Corneas were kept in organ culture for 12 hours thereafter. To evaluate cell-mediated immune reaction, immunofluorescent staining for leucocytes (cluster of differentiation 45) and specifically for dendritic cells (human leukocyte antigen-DR) was performed in every group. A terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was used to determine apoptosis reaction. RESULTS: The ratio of dendritic cells in the femtosecond group compared with the microkeratome group was 1.2 (P=.02), the ratio of leucocytes was 1.4 (P=.06), and the ratio of apoptotic cells was 1.0 (P=.59). There was no marked significant difference in the distribution of inflammatory cell reaction. The control group showed neither specific inflammatory reaction nor apoptosis. CONCLUSION: This in vitro series of human corneas showed similar inflammatory tissue reaction after femtosecond laser-assisted and microkeratome-assisted flap creation (P<.05). FINANCIAL DISCLOSURE: No author has a financial or proprietary interest in any material or method mentioned.

Optimizing the deswelling process of organ-cultured corneas.

PURPOSE: During deswelling of organ-cultured human corneas, endothelial cell loss occurs. Therefore, it is necessary to minimize the deswelling time and achieving an optimal central corneal thickness (CCT) of approximately 550 microm at the same time. We investigated the minimal deswelling time necessary and analyzed endothelial cell loss. METHODS: Fifty-eight human corneas were stored between 13 and 81 days in organ culture. CCT was measured by optical coherence tomography. Measurements were performed before preparation, during culturing, before deswelling, and after varying deswelling periods (1-72 hours) using 5% dextran. Additionally, vital staining was performed in 6 human corneas to assess endothelial cell loss between 24 and 30 hours of deswelling. To evaluate absolute cell loss, endothelial cells were counted on human corneal pairs after 24 and 30 hours of deswelling. RESULTS: After organ culture, mean CCT was 1194 microm. After 24 hours of deswelling in dextran-containing medium, mean CCT was 600 microm, whereas after 30 hours, mean CCT was 510 microm and hardly any corneas showed a CCT of more than 550 microm. Almost no further decrease in CCT was observed thereafter. No factors could be identified predicting the necessary deswelling time; however, paired corneas showed significant correlation of deswelling characteristics. We did not see any differences in endothelial cell loss 24 and 30 hours of deswelling or the ratio of living to dead endothelial cell counts. CONCLUSIONS: Deswelling for 24 hours does not provide an optimal corneal thickness. Because endothelial cell loss does not increase between 24 and 30 hours of deswelling, a period of 30 hours is more suitable for obtaining sufficient corneal thickness.

Ocular manifestation of primary VZV infection in a splenectomized patient.

PURPOSE: To report a rare case of primary varicella zoster virus (VZV)-associated retinal vasculitis in a splenectomized patient. DESIGN: Case report. RESULTS: After manifestation of VZV-associated retinal vasculitis, a splenectomized patient experienced binocular loss of vision. CONCLUSIONS: For the development of VZV-associated uveitis, the presence of specific T cells are necessary. Here, the authors present a rare case of VZV-associated retinal vasculitis in a splenectomized patient.

The protective effect of quercetin against oxidative stress in the human RPE in vitro.

PURPOSE: To investigate the possible protective effect of the dietary antioxidant quercetin on retinal pigment epithelial (RPE) cell dysfunction and cellular senescence occurring in age-related macular degeneration (AMD). The major flavonoid quercetin was studied on RPE cells in vitro. METHODS: Cultured human RPE cells were incubated with different concentrations of quercetin for 24 hours. Cells were then treated with 150 to 300 microM hydrogen peroxide for 2 hours. Mitochondrial function was measured by using MTT assay and cell vitality by live-dead staining assay. Intracellular levels of glutathione were determined by using a glutathione assay kit. Apoptosis was quantified by a caspase-3 assay, and cellular senescence was quantified by beta-galactosidase staining. Expression of the senescence-associated transmembrane protein caveolin-1 was investigated by Northern and Western blot analyses. RESULTS: Hydrogen peroxide treatment caused significant decreases in mitochondrial function (52%) and in cell vitality (71%), whereas preincubation with 50 microM quercetin diminished this decrease in a dose-dependent manner. Quercetin treatment did not show any notable effect on intracellular levels of glutathione in either used concentration of quercetin. Hydrogen peroxide-induced activation of caspase-3 was reduced by 50 microM quercetin, from 1.9- to 1.4-fold, compared with untreated control (P < 0.001). Hydrogen peroxide caused a large (>90%) dose-dependent increase in beta-galactosidase-positive cells, whereas in the untreated control only single cells expressed this enzyme (<5%). This increase in cellular senescence was significantly attenuated by quercetin in a dose-dependent manner. The highest attenuation was reached at 50 microM quercetin. Quercetin caused a significant dose-dependent reduction of caveolin-1 mRNA 48 hours after treatment with hydrogen peroxide. After 96 hours of incubation, caveolin-1 protein levels were also reduced. CONCLUSIONS: The data demonstrate that quercetin is able to protect RPE cells from oxidative damage and cellular senescence in vitro in a dose-dependent manner. The authors suggest that this increase in antioxidative capacity is--among other mechanisms, such as the intracellular redox state--also mediated by inhibiting the upregulation of caveolin-1. Downregulation of caveolin-1 may be important for the retinal pigment epithelium to prevent apoptotic cell death in response to cellular stress, a condition implicated in the early pathogenesis of AMD. Therefore, the authors believe that the use of antioxidative dietary flavonoids such as quercetin is a promising approach in the prevention of early AMD.

Intravitreal bevacizumab injections for treatment of central retinal vein occlusion: six-month results of a prospective trial.

PURPOSE: To evaluate the effect of intravitreal bevacizumab (Avastin; Genentech, Inc., South San Francisco, CA) injections on visual acuity and foveal retinal thickness in patients with central retinal vein occlusion (CRVO). METHODS: In this prospective, noncomparative, consecutive, interventional case series, 46 patients received repeated intravitreal injections (1.25 mg) of bevacizumab. Main outcome measures were visual acuity (Snellen and ETDRS charts) and optical coherence tomography measurements in a 6-month follow-up period. RESULTS: Mean visual acuity improved from 20/250 at baseline to 20/80 at the 6-month follow-up (P < 0.001). ETDRS chart findings revealed a mean letter gain +/-SD from baseline to 6 months of 13.9 +/- 14.4 letters. Mean central retinal thickness +/-SD decreased from 535 +/- 148 microm at baseline to 323 +/- 116 microm at the 6-month follow-up. Ischemic CRVO was associated with significantly lower visual acuity than nonischemic CRVO (P < 0.001). However, visual acuity gain was similar in both groups. Independent of duration of symptoms, CRVO was associated with a similar gain in visual acuity. CONCLUSION: Intravitreal injection of bevacizumab appears to be a new treatment option for patients with macular edema secondary to CRVO.

Detection of laser in situ keratomileusis in a postmortem eye using optical coherence tomography.

With more individuals having laser in situ keratomileusis (LASIK), eye banks are challenged to detect prior refractive surgery in donor tissue. We report the case of a donor who had LASIK 9 months before his death. Slitlamp biomicroscopy, corneal topography, and optical coherence tomography (OCT) were performed to evaluate the corneas. Few changes were detected under slitlamp examination and corneal topography. We demonstrate that OCT is capable of detecting LASIK-induced structural changes in the immediate postmortem evaluation and during the early and late period of organ culture. We recommend OCT screening of potential donor corneas before organ culture and between days 9 and 12 of organ culture.

Optical coherence tomography for the detection of laser in situ keratomileusis in donor corneas.

PURPOSE: In 2001, more than one million laser in situ keratomileusis (LASIK) procedures were performed worldwide. Considering the increasing number of refractive procedures, eye banks will be increasingly confronted with the problem of how to identify those donors with prior refractive surgery. To date, efficient screening methods to identify LASIK surgery in donor eyes have not been established. Therefore, the purpose of the current study was to determine whether optical coherence tomography (OCT) can be used to detect the presence of LASIK-induced changes in human corneas. METHODS: Laser in situ keratomileusis was performed on 20 organ-cultured human cornea disks. The excimer laser ablation performed ranged from 0 to 12 diopters. The corneas were maintained in culture, and the visibility of flap-stromal interface by OCT was assessed up to 6 months after the LASIK procedure. Additionally, two donor corneas with the history of LASIK treatment before death were screened for structural changes. RESULTS: Optical coherence tomography scans were able to detect the interface between the corneal flap and the residual stromal tissue in all corneas and at all examined time intervals. There were no differences in signal intensity among the different depths of ablation. The relative signal intensity of the interface compared with the averaged stromal intensity ranged from 2.1 to 6.0. In both donor corneas with suspected prior LASIK surgery, OCT scanning showed the characteristic stromal interface as found in the in vitro model. CONCLUSIONS: Corneal examination by OCT could be an appropriate technique for eye banks to screen donor corneas for prior LASIK surgery.