Latent endothelial cell damage after experimental corneal cryopreservation

Ninety porcine corneas were evaluated by vital staining with alizarin red S and trypan blue in a three-step experiment. Central cell densities were counted (a) on freshly dissected corneas (n = 30), (b) on cryopreserved corneas directly after thawing (n = 30), and (c) after a postthawing organ culture interval of 24 h (n = 30). Two freezing methods were used: (a) minimum essential medium--containing 20% fetal calf serum and (b) the same but containing additionally 2% chondroitin sulfate. Directly after thawing neither method showed significant cell loss (3.9% and 3%) compared to fresh tissue. After postthawing organ culture, however, tissue that had been frozen without chondroitin sulfate displayed a cell loss of 73.5% compared to corneas of the same freezing protocol directly after thawing. Corneas in chondroitin sulfate containing medium showed a cell loss of only 33.2%. We conclude that reliable morphologic evaluation should not be obtained from cryopreserved corneas examined directly after thawing.     

Informing the patient

PURPOSE: To investigate the feasibility to use hydroxyethylstarch as an alternative deswelling additive in short-term preservation media. MATERIALS AND METHODS: Corneoscleral discs were prepared from pairs of eye balls of freshly slaughtered pigs. Corneas were stored in MEM-medium containing either 10% or 20% hydroxyethylstarch 450 000 at 4 degrees C in a refrigerator. Subsequently, the tissue was stored for 24 hours in organ culture at 37 degrees C in MEM-medium containing 10% fetal calf serum to detect latent endothelial cell damage. Mate corneas were treated the same except for being stored in Optisol GS during 4 degrees C storage. We determined corneal endothelial cell density, stromal thickness, and glucose concentration in the medium directly after preparation, after short-term storage at 4 degrees C, and after subsequent organ culture at 37 degrees C. Scanning electron microscopy of corneal endothelium was performed at each step during the experimental course. RESULTS: We did not observe any significant differences in endothelial-cell density between experimental groups and control groups. No decrease in endothelial-cell density was observed during the course of experiments. No increase in stromal thickness was determined in any group after short-term storage at 4 degrees C. Corneas stored in medium containing 20% hydroxyethylstarch showed a decrease in stromal thickness after short-term storage. After subsequent organ culture all corneas displayed a uniform stromal swelling. Glucose concentrations in the media decreased in all groups during the experiment. In scanning-electron microscopy we observed a reversible degeneration of cell borders after storage at 4 degrees C. Additionally, corneas stored in Optisol GS showed a reversible cobblestone appearance at this stage of the experiments. CONCLUSION: Hydroxyethylstarch appears to be an alternative to the use of dextran and chondroitin sulfate as a deswelling additive in corneal preservation media.     

Phorbol ester modulation of rabbit corneal endothelial permeability

PURPOSE: Phorbol esters have been shown to have a profound influence on cellular activity in many cell types. The purpose of this study was to examine the influence of phorbol esters on the function and structure of corneal endothelial cells. METHODS: Corneas were placed under a specular microscope, and the endothelium was superfused with glutathione bicarbonate Ringer's solution (GBR); with GBR and 10 nM, 100 nM, or 1 microM 4 beta-phorbol 12-myristate 13-acetate (PMA); or with 100 nM 4-alpha-PMA. Corneal swelling curves were generated, and endothelial permeability was determined. Corneal endothelial structure was examined with a scanning electron microscope. RESULTS: Significant increases in swelling and endothelial permeability were found in corneas perfused with 100 nM PMA versus that observed in controls (swelling rate = 26 microns/hr versus 6.9 microns/hr; permeability = 6 x 10(-4) cm/min versus 3.4 x 10(-4) cm/min) and in corneas receiving 1 microM PMA versus that in controls (swelling rate = 26.3 microns/hr versus 0.12 micron/hr; permeability = 6.9 x 10(-4) cm/min versus 4.9 x 10(-4) cm/min). Application of 10 nM PMA did not significantly alter either parameter. Study with transmission electron microscope demonstrated significant morphologic changes in cells perfused with all concentrations of PMA. Corneas perfused with 100 nM 4-alpha-PMA versus 100 nM PMA had significantly lower slope and permeability values (swelling rate = 5.9 microns/hr versus 25.1 microns/hr; permeability = 3 x 10(-4) cm/min versus 6.7 x 10(-4) cm/min). CONCLUSIONS: Phorbol esters are detrimental for corneal endothelial function, creating significant corneal swelling, increases in endothelial permeability, and changes in endothelial cell structure. This effect appears to be mediated through a protein kinase C pathway.     

Assessment of cornea viability by confocal laser scanning microscopy and MTT assay

PURPOSE: Determination of excised cornea viability is of interest for transplant-storage evaluation, but also for in vitro diffusion-study design and ocular-toxicity assessment. By using simultaneous vital staining by calcein AM (CAM) and ethidium homodimer-1 (EH-1), as "live" and "dead" probes, respectively, we developed a confocal laser scanning microscopy (CLSM) assay to determine epithelial and endothelial viability and estimate cornea thickness. METHODS: New Zealand White rabbit corneas were stored in phosphate-buffered saline (PBS) or Optisol at 4 degrees C or at room temperature. At various times, corneas were stained with an EH-1/CAM solution and observed, without further treatment, by CLSM. Storage effects on the cornea were also assessed by using an MTT assay. RESULTS: Stromal swelling, shedding of the upper epithelial layers, and severe endothelial damage were observed after 4 h in PBS at room temperature. After 8 h, lower epithelial cell death was observed, along with loss of endothelial structure. Corneas stored in similar conditions in Optisol were indistinguishable from controls. Storage in Optisol at 4 degrees C affected the superficial layers of the corneal epithelium similarly at both 7 and 14 days. Extensive epithelial shedding and wing-cell death were observed at 25 days, but the basal layer remained approximately 50% healthy. Significant endothelial cell loss was observed at 25 days. MTT results were consistent with CLSM data in the medium-term storage study only. CONCLUSIONS: This CAM/EH-1 CLSM fluorescence assay is a sensitive index of viability in cornea, and thus may prove useful in investigations in which maintenance of vital functions in different cell layers is critical.     

Expression of proliferating cell nuclear antigen in corneas kept in long term culture

PURPOSE: To investigate the proliferative activity of the donor corneal cells and to examine how this property changed during long term culture. METHOD: Fourteen human corneas from donors (ages from 50-91) were cultured in the medium (MEM+8% FBS with or without dextran). The proliferating status of corneal cells was evaluated by immunohistochemical staining of proliferating cell nuclear antigen (PCNA) in the cells. Three corneas at each time point were fixed in paraformalin at day 0, day 3 and after 3 weeks cultured in medium as well as 3 weeks plus 2 or 5 days in fresh medium with 8% dextran. Paraffin-embedded corneas were sectioned to 4 microm and stained with antibody PC 10 against PCNA. The number of PCNA positive cells was identified under light microscope. RESULT: Prior to organ culture only basal limbal epithelial cells stained positive for PCNA. After 3 days in culture 50 percent of the epithelial cells were positive as were several keratocytes and some endothelial cells in the peripheral corneas. After 21 days no cells showed proliferative activity. After 21 days in culture and 5 days in fresh deswelling medium the essentially monolayered epithelium stained positively in the limbal area. The proliferative activity of the keratocytes in the anterior stroma was extensive. Endothelial cells stained positive in the peripheral cornea. CONCLUSION: Limbal epithelial cells appear to survive in the organ culture. The corneas may be worth evaluating as sources of stem cells for grafting. Likewise, the keratocytes survive organ culture and can be induced to proliferate after a change to fresh medium. The endothelium is stimulated to proliferate in organ culture and in fresh medium after long term storage.     

Effect of surface photorefractive keratectomy and laser in situ keratomileusis on the corneal endothelium

PURPOSE: To investigate endothelial cell loss in pairs of fresh human autopsy globes following high-diopter myopic photorefractive keratectomy (PRK) or laser in situ keratomileusis (LASIK). SETTING: Center for Research on Ocular Therapeutics and Biodevices and Magill Laser Center for Vision Correction, Storm Eye Institute, Charleston, South Carolina, USA. METHODS: In the first part of the study, 12 globes had either -10 diopters (D) multizone surface PRK or -10 D single-zone LASIK. In the second part, three groups of 5 globes each had -15 D, -20 D, or -25 D multizone-blend LASIK procedures. Fellow globes in both groups were used as untreated controls. Corneoscleral buttons were excised from all globes. Following 7 days in corneal organ culture, the endothelial surface was stained with two vital dyes: calcein-AM and ethidium homodimer. Fluorescence microscopy was used to obtain endothelial cell counts. RESULTS: The mean dead cells per square millimeter (cells/mm2) were 0.94 in the -10 D PRK treated corneas compared with 0.91 in the fellow untreated eyes (P = 0.06(. The mean dead cells/mm2 in the -10 D single-zone LASIK-treated corneas and in the fellow untreated eyes were 0.61 (P = 0.88). The mean dead cells/mm2 in the -15 D, -20 D, and -25 D multizone-blend LASIK-treated corneas were 3.08, 2.33, and 5.55, respectively, compared with 3.49, 1.92, and 5.01 in the fellow untreated eyes (P = 0.276, P = 0.339, and P = 0.427, respectively). Dead cell counts for treated and control paired corneas were highly correlated in all treatment groups. CONCLUSIONS: No significant endothelial cell loss occurred after -10 D PRK or LASIK corrections up to -25 D. Although this study has limitations that prevent direct extrapolation to the clinical situation, it does afford a comparable clinical correlate for endothelial cell toxicity following a typical excimer laser ablations.     

Laser skin resurfacing. Er:YAG laser and cw-CO2 laser with scanner system in direct comparison]

PURPOSE: To integrate a newly developed OLCR instrument into the optical system of the excimer laser. The instrument is designed to perform corneal pachymetry before, during, and after corneal photoablation and thus allow for a precise and continuous on-line measurement of the corneal photoablation process. METHODS: The conditions required to integrate the OLCR instrument into the excimer laser optics were investigated. With a technical setting providing on-line data of corneal thickness, three groups of 8-10 corneae received central keratectomies of 27 (group 1), 82 (group 2) and 163 (group 3) microns calculated central depth and 7.38 mm diameter. All measurements were performed with OLCR and ultrasound. RESULTS: The OLCR instrument was coupled into the optical system of the excimer laser and a useful signal obtained at SLD power levels of 40 microW incident on the cornea. Individual corneal thickness measurements were obtained before, during and after the photoablation procedure. In group 1, the ablation was 50.3 (40-68) microns measured with ultrasound and 30.2 (27-38) microns measured with OLCR. In group 2, the ablation was 101.1 (80-113) microns measured with ultrasound and 93.3 (76-109) microns measured with OLCR. In group 3, the ablation was 210.6 (190-227) microns measured with ultrasound and 188.4 (181-197) microns measured with OLCR. The precision (standard deviation) for measurements of individual corneas was 1-2 microns with OLCR and up to 12 mm in Ultrasound measurements. CONCLUSION: With this interferometric method, continuous, non-contact measurement of corneal thickness before, during and after excimer laser photoablation were performed. By establishing a feed-back control between the pachymetric measurements and the photoablation process, the precision of excimer ablation may possibly be further increased.     

Experimental erbium: YAG laser photoablation of trabecular meshwork in rabbits: an in-vivo study

Photoablative laser trabecular surgery has been proposed as an outflow-enhancing treatment for open-angle glaucoma. The aim of the study was to investigate the time course of repair response following low-thermal Erbium: YAG laser trabecular ablation. In 20 anaesthetized rabbits gonioscopically controlled ab-interno photoablation of the ligamenta pectinata and underlying trabecular meshwork (TM) was performed with a single-pulsed (200 microseconds) Erbium: YAG (2.94 microns) laser. The right eye received 12-15 single laser pulses (2 mJ) delivered through an articulated zirconium fluoride fiberoptic and a 200 microns (core diameter) quartz fiber tip, the left unoperated eye served as control. At time intervals of 30 minutes, 2, 10, 30, and 60 days after laser treatment, eyes were processed for light- and scanning electron microscopy. The applied energy density of 6-4 J cm-2 resulted in visible dissection of the ligamenta pectinata and reproducible microperforations of the TM exposing scleral tissue accompanied by blood reflux from the aqueous plexus. The initial ablation zones measured 154 +/- 36 microns in depth and 45 +/- 6 microns in width. Collateral thermal damage zones were 22 +/- 8 microns. At two days post-operative, ablation craters were still blood- and fibrin-filled. The inner surface of the craters were covered with granulocytes. No cellular infiltration of the collateral thermal damage zone was observed. At 10 days post-operative, progressive fibroblastic proliferation was observed, resulting in dense scar tissue formation with anterior synechiae, proliferating capillaries and loss of intertrabecular spaces inside the range of former laser treatment at 60 days post-operative. Trabecular microperforations were closed 60 days after laser treatment in all rabbits. IOP in treated and contralateral eyes did not significantly change its level during whole period of observation. Low-thermal infrared laser energy with minimal thermal damage to collateral structures could not effectively prevent early scarring of trabecular surgery in rabbits.     

Carbon dioxide laser-assisted hair transplantation. The effect of laser parameters on scalp tissue--a histologic study

BACKGROUND: "Char-free" carbon dioxide lasers are capable of precise tissue vaporization with minimum residual thermal damage. These lasers deliver very high bursts of energy for extremely short durations, and can vaporize recipient sites during hair transplantation, which are consistent in depth and diameter. OBJECTIVE: To determine the depth of ablation and lateral thermal damage of scalp tissue using various laser parameters with the Sharplan 80XJ Silklaser. METHODS: Two hundred forty laser recipient sites were created, using 24 different laser parameters in each of 10 specimens of scalp tissue removed during scalp reduction. These specimens were evaluated histologically for depth of ablation and lateral thermal damage. RESULTS: While some laser parameters worked better regarding consistent depth of ablation of scalp tissue, lateral thermal damage was quite narrow (average, 20-50 microns). CONCLUSIONS: None of the laser parameters tested in this study should be problematic when used clinically for hair transplantation.     

Serum-free media for culturing and serial-passaging of adult human retinal pigment epithelium

The ability of a chemically-defined serum-free culture medium to support the attachment, growth and serial passaging of primary adult human retinal pigment epithelial (RPE) cells was studied. Primary cultures of adult human RPE were established in a chemically-defined serum-free culture medium on both bare or bovine corneal endothelial extracellular matrix-coated tissue-culture plastic. Confluent cells were serially passaged in chemically-defined serum-free culture medium three times by trypsinization, and trypsin activity was quenched with aprotinin. First passage RPE cells were plated onto tissue-culture plastic precoated with bovine corneal endothelial extracellular matrix or uncoated tissue-culture plastic in 24 well plates at a density of 50 viable cells mm-2. Cells were maintained either in chemically-defined serum-free culture medium, DMEM without serum, or DMEM with 15% fetal bovine serum. For each medium plating, efficiencies were determined 24 hours after plating, and growth rates were determined on the first, third and seventh days after plating. Morphometric image analysis was performed on cells cultured for up to 6 weeks and three serial passages. Seeding efficiency on bovine corneal endothelial extracellular matrix-coated tissue-culture plastic and treated tissue-culture plastic were higher for chemically-defined serum-free culture medium (88.9+/-2.7% and 47.1+/-4.1%, respectively) and DMEM with serum (87.2+/-5.6% and 52.9+/-10.5%, respectively) than DMEM without serum (59.2+/-5.6% and 33.1+/-6.9%, respectively; P<0.01). The RPE proliferation rate in chemically-defined serum-free culture medium was comparable to DMEM with serum on both substrates within the first 3 days, although cells in DMEM with serum had a higher proliferation rate on day 7. Cells cultured in DMEM without serum, eventually decreased in number. RPE maintained in chemically-defined serum-free culture medium maintained a consistent proliferation rate, reached confluence, and retained an epitheloid morphology on either extracellular matrix or tissue-culture plastic for up to 6 weeks and three serial passages. Primary RPE reached confluence at 12+/-3 days on bovine corneal endothelial extracellular matrix-coated tissue-culture plastic and 21+/-5 days on treated tissue-culture plastic. Confluent cultures were composed of small hexagonal cells with epitheloid morphology on both substrates. We concluded that primary adult human RPE can be cultured in this chemically-defined serum-free culture medium. RPE will proliferate, reach confluence, retain their epitheloid morphology and can be serially passaged in the absence of serum.     

Optimizing the review process of food additive and GRAS petitions. Overview of the workshop

PURPOSE: To assess endothelial barrier function, morphological appearance and corneal thickness three months after cataract surgery in order to evaluate intraoperative endothelial damage. METHODS: Endothelial permeability was examined by fluorophotometry, and contact specular microscopy and corneal pachymetry measurements were made in 40 patients (40 eyes) with senile, non-complicated cataracts one month before and three months after cataract surgery. Twenty eyes underwent uneventful phacoemulsification (Group 1) and 20 uneventful extracapsular cataract extraction (ECCE) with continuous curvilinear capsulotomy (Group 2). Results were analyzed using the two-tailed Student's t test, analysis of variance, and multifactorial and regression analysis. RESULTS: There was a significant postoperative increase in endothelial permeability in both groups (p < 0.001), but no real differences between the postoperative values (p = 0.07). Mean cell loss was 15.2% in ECCE and 18.3% in phacoemulsification (p = 0.4). There was a significant linear correlation between ultrasound time, cell loss and functional damage. Postoperative pachymetric measurements were not significant. CONCLUSIONS: Endothelial response showed no differences between the surgical techniques. Endothelial barrier function remained disturbed in spite of the apparent morphological stabilization. Corneal pachymetry is not useful for assessing postoperative endothelial changes.     

Skin resurfacing of facial rhytides and scars with the 90-microsecond short pulse CO2 laser. Comparison to the 900-microsecond dwell time CO2 lasers and clinical experience

BACKGROUND: Carbon dioxide lasers that produce either short pulses or scanned continuous beams have been used for skin resurfacing to improve wrinkles or scars. Using a high peak power, short pulse CO2 laser can produce clinically effective results with minimal thermal damage. OBJECTIVE: To evaluate the effectiveness of skin resurfacing using the 90-microsecond pulse duration CO2 laser compared to other laser systems. Erythema, healing time, complications, and histological measurement of the depth of ablation and thermal damage per pass with this system were also assessed. METHODS: Forty-one patients with facial rhytides and scars underwent resurfacing with a 90 microseconds pulse duration CO2 laser. Using patient survey, patients were evaluated for effectiveness of therapy, healing time, and complication rates. Comparisons of histologic and clinical findings were made with different short pulse CO2 lasers. RESULTS: Healing time, duration of erythema, and post-operative pain were less with the 90 microseconds pulse CO2 laser than with the 900-microsecond dwell time and 950-microsecond pulse duration lasers, while effectiveness was comparable. Complications were few with the 90-microsecond pulse laser, including three patients (9.1%) developing hyperpigmentation. One pass with the 90-microsecond pulse duration CO2 laser produced 100 microns of ablation with 17 microns of thermal damage. Ablation and damage were additive so that, by six passes, ablation depth was 350 microns and depth of thermal damage was 63 microns. This thermal damage is less than that reported with lasers having a longer pulse duration or dwell time with comparable depths of vaporization. CONCLUSION: Treatment with the 90-microsecond pulse duration laser results in a more rapid healing time and shorter duration erythema. The clinical improvements in wrinkles and sun damage were comparable. The 90-microsecond pulse duration laser provides an effective, predictable, and safe means of improving facial rhytides and scars.     

Properties of cochlear nucleus neurons in primary culture

Dissociated primary cell cultures were derived from the cochlear nuclei (CN) of postnatal rats using standard techniques. Cultured cells differentiated morphologically, but their dendritic profiles were generally less specialized than those of CN cells in vivo. Physiologically, cultured cells could be divided into three classes: tonic, phasic and non-spiking cells, which differed in many of their fundamental biophysical properties. The percentage of cultured cells that spiked repetitively increased over time to a maximum of 85% at 6 days. However, the percentage of cells that produced action potentials decreased with time in culture, from 91% during the first 8 days to less than 40% after 9 days. CN cells were successfully cultured in both serum-supplemented and serum-free (Neurobasal) media. More neurons survived at low plating densities in Neurobasal than in medium containing serum, although neuronal survival was similar at higher densities. Few neurons raised in the serum-free medium were spontaneously active; other response properties were similar to those of cells grown in the presence of serum. Although differentiation of CN cells in culture did not completely mirror the in vivo developmental pattern, these experiments demonstrate that primary culture represents a viable method for the in vitro study of CN neurons.     

Specific activity of polypyrrole nanoparticulate immunoreagents: comparison of surface chemistry and immobilization options

PURPOSE: Angioscopy for in situ vein graft preparation has been criticized on the basis that the trauma of instrumentation may predispose to accelerated intimal hyperplasia, jeopardizing patency rates following infrainguinal revascularization. The aim of this study was to assess the effects of angioscopic preparation on endothelial integrity and smooth muscle cell (SMC) behavior in an established organ culture model of human saphenous vein (HSV). METHODS: HSV was harvested from 12 patients during bypass surgery before and after angioscopic preparation. Endothelial integrity was evaluated by immunohistochemical staining with JC-70 and scanning electron microscopy (SEM); remaining segments of pre- and postangioscopy vein were maintained in culture for 14 days in medium supplemented with 30% fetal calf serum. Viability was confirmed by measurement of tissue adenosine triphosphate on day 14 and thickness of the neointima was measured by computerized image analysis of histologic sections. Monoclonal antibodies to proliferating cell nuclear antigen (PCNA) were used as an immunohistochemical marker for proliferating SMCs. RESULTS: There was a significant reduction in the percentage staining by JC-70 (71.3% versus 20.4%) in pre- versus postangioscopy vein (p = 0.002 by Wilcoxon's rank test; n = 12). This was supported by SEM images. Despite this, there were no significant differences between the pre- and postangioscopy HSVs after 14 days of culture with respect to neointimal thickness (61 versus 56 microns) and staining with PCNA (4.80 versus 4.08 nuclei per 10 microns), all according to Wilcoxon's rank test. CONCLUSIONS: Angioscopic vein graft preparation is associated with endothelial cell loss but does not induce additional neointimal hyperplasia in HSV in vitro. These results suggest that angioscopic manipulation does not alter SMC behavior.     

Human corneal studies with a vitrification solution containing dimethyl sulfoxide, formamide, and 1,2-propanediol

We tested the tolerance of human corneas to a vitrification solution, modified VS41A, containing 3.1 M dimethyl sulfoxide, 3.1 M formamide, and 2.2 M 1,2-propanediol in a carrier solution consisting of the corneal storage medium CPTES with 2.5% w/v chondroitin sulfate. Seven human corneas were exposed for 10 min each to graded concentrations of the solution at 0 degree C, remaining in the full-strength solution for 10 min. The corneas had significantly more endothelial cell damage (P < 0.05) than seven mated control corneas, but it was minimal (4.3% cell loss). Attempts at vitrification and rewarming of three corneas exposed to the solution by this protocol, however, resulted in ice formation in the peripheral corneal stroma and severe endothelial damage. Presumably, equilibration with the cryoprotectant in the thicker periphery of the human cornea had not occurred. Ice did not form on the center of one cornea, and substantial numbers of central endothelial cells survived after vitrification in this case. Immersion of the human corneas for 25 min in each of the four graded solutions at 0 degree C was required for sufficient penetration of the cryoprotectant to allow total corneal vitrification and rewarming without ice formation. This prolonged exposure to modified VS41A caused unacceptable damage to the corneal endothelium, however. Successful vitrification of human corneas with this solution will require a safe method for obtaining corneal equilibration with the cryoprotectant.     

Hidradenitis suppurativa of the groin, treated by excision and spontaneous healing

We examined and photographed the central corneal endothelium of 16 patients with the clinical specular microscope before and at intervals after cataract extraction. No detectable loss of the endothelial cells occurred in 75% of the patients, including 12 routine intracapsular cryoextractions and four phacoemulsifications of soft cataracts in young adults. Only one of the four cases of significant endothelial cell loss occurred in a normal cornea without demonstrable operative or postoperative complications. Two of the four corneas that lost central endothelial cells at cataract extraction continued to lose more cells during the ensuing weeks. A significant increase in central endothelial cell density was demonstrated in one patient. The four corneas with endothelial cell loss also had a significantly higher mean increase in corneal thickness postoperatively, although this was transient. All 16 corneas remained clear during the period of observation (maximum, 14 weeks), and long-term studies are needed to measure the chronic effects of the endothelial damage.     

Alcohol versus mechanical epithelial debridement: effect on underlying cornea before excimer laser surgery

PURPOSE: To determine the effects of 70% isopropyl alcohol used for corneal debridement on surface smoothness, stromal keratocytes, and ease of epithelial removal. SETTING: Cornea Research Laboratory, University of Rochester, and Excimer Laser Laboratory, Genesee Valley Eye Institute, Rochester, New York, USA. METHODS: Rabbit corneas were de-epithelialized mechanically or with 70% alcohol. The rabbits were split into groups and evaluated immediately or after a 50 microns deep excimer laser phototherapeutic keratectomy. All tissue was evaluated and compared in terms of surface smoothness parameters, loss of keratocytes, and inflammatory response to de-epithelialization. RESULTS: Computerized laser interferometric microscopy showed no between-group difference in the surface smoothness parameters. There was a marked absence of keratocytes in the superficial 25% of the corneal stroma. The loss of keratocytes was significantly higher (P < .001) in corneas treated with isopropyl alcohol. The inflammatory response 24 hours after epithelial removal was significantly higher (P < .001) in the corneas treated with alcohol. CONCLUSION: The use of 70% isopropyl alcohol applied for 2 minutes for epithelial removal did not enhance the quality of the subsequent excimer laser procedure. In contrast, isopropyl alcohol increased the inflammatory response, and it may have damaging effects on keratocytes. We would not advocate the use of 70% isopropyl alcohol as administered in our study to remove corneal epithelium before excimer laser surgery.     

Effects of the pharmaceutical cosolvent hydroxypropyl-beta-cyclodextrin on porcine corneal endothelium

BACKGROUND: The influence of the pharmaceutical cosolvent hydroxypropyl-beta-cyclodextrin (HPBCD) on porcine corneal endothelium was investigated. The purpose was to find out if this substance causes severe damage to the cornea. METHODS; One hundred and ninety-five pig corneas were preserved in Eagle's minimal essential medium with dextran and HPBCD. They were stained with trypan blue, examined using a light microscope and then reincubated. Changes in cell density and cell morphology as a function of the duration of preservation and the HPBCD concentration were evaluated. We developed a morphological classification combining the morphological aspects observed using the light microscope and the scanning electron microscope. The vitality of the endothelium was analyzed by cell separation and monolayer cultivation. RESULTS: The cell density stayed stable without significant alterations in 0.1% HPBCD, 1% HPBDC and control solutions. In 10% HPBDC, however, the endothelium showed significant loss of cells. The morphological classification revealed high-grade endothelial damage in 10% HPBDC and low-grade damage in 1% HPBCD. The changes observed in 0.1% HPBCD and control medium were comparable. The degree of alteration conformed to the results of monolayer cultivation: endothelial cells of damaged corneoscleral buttons were limited in their ability to proliferate. CONCLUSION: Severe endothelial destruction in 10% HPBCD and changes in membrane integrity at lower concentrations limit the use of HPBCD in ophthalmic solutions.     

Toxicity of Xylocaine to rabbit corneal endothelium

PURPOSE: To assess the toxicity of lidocaine hydrochloride (Xylocaine) to the corneal endothelium. SETTING: Department of Ophthalmology, H?tel-Dieu Hospital, Paris, France. METHODS: Rabbit corneas were excised and the endothelium was exposed to balanced salt solution (BSS), Xylocaine 1%, or Xylocaine 5% (5 corneas/group) for 20 minutes. The endothelium was then stained with trypan blue and alizarin red, and 5 photomicrographs were taken of each cornea at a standard magnification and analyzed with a digital imaging system (Biocom 200). RESULTS: Xylocaine solutions produced changes in endothelial cell morphology, but there was no cell staining with trypan blue. Corneas exposed to Xylocaine 5% had more marked cell alterations. Small areas of cells were lost from all 15 corneas, mainly at the periphery, but the differences among the 3 groups of corneas were not significant. CONCLUSION: Exposure of rabbit corneal endothelium to Xylocaine solutions in vitro was not associated with trypan blue staining of endothelial cells.     

A double-masked, randomized, 1-year study comparing the corneal effects of dorzolamide, timolol, and betaxolol. Dorzolamide Corneal Effects Study Group

OBJECTIVE: To compare the long-term effects of dorzolamide hydrochloride (Trusopt, Merck and Co Inc, White-house Station, NJ), timolol maleate, and betaxolol hydrochloride on corneal endothelial cell density and corneal thickness. METHODS: This 1-year multicenter study was conducted in 298 patients with ocular hypertension or open-angle glaucoma who had a baseline central corneal endothelial cell density greater than 1500 cells/mm2 and central corneal thickness less than 0.68 mm in each eye. Patients were randomized to 0.5% betaxolol twice daily, 0.5% timolol twice daily, or 2.0% dorzolamide 3 times daily. Specular microscopy and ultrasonic pachymetry of the central cornea was performed at baseline and 6 and 12 months following institution of therapy. Endothelial cell densities were determined by a single masked observer. RESULTS: The mean percent changes from baseline for both outcome measures were similar in all 3 treatment groups at both 6 and 12 months. After 1 year of treatment, the mean percent loss in endothelial cell density from baseline was 3.6%, 4.5%, and 4.2% for the dorzolamide, timolol, and betaxolol groups, respectively. The mean percent change from baseline for corneal thickness was 0.47%, -0.25%, and 0.39% for the dorzolamide, timolol, and betaxolol groups, respectively. CONCLUSIONS: Dorzolamide is equivalent to timolol and betaxolol in terms of the change in central endothelial cell density and thickness after 1 year of therapy. All 3 treatments exhibit good long-term corneal tolerability in patients with normal corneas at baseline.