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.     

Duration of fluorescein diacetate in corneal endothelium

A new optical measurement technique based on spectral scanning (the scanning Soret oximeter) has previously been described. The technique has now been tested under extremes of physiological pH and temperature, the effects of which are shown to be negligible.     

Mitotic inhibition of corneal endothelium in neonatal rats

PURPOSE: Corneal endothelium in humans does not divide to any significant extent after birth; therefore, with age there is a gradual loss of cells. When cell density is reduced to a critical level, the endothelium cannot function to maintain corneal clarity, and the cornea becomes permanently cloudy. Currently, the blindness that results can be treated only by corneal transplantation. The long-term goal is to find methods to stimulate corneal endothelial proliferation in a clinically relevant manner. The first step toward achieving this goal is to identify mechanisms responsible for the induction and maintenance of mitotic inhibition of the corneal endothelium in vivo. During corneal development, the endothelium is formed by migration and proliferation of mesenchymal cells from the ocular periphery. Soon after the monolayer is formed, proliferation ceases. In tissue culture, many cell types cease proliferating upon formation of stable cell-cell and cell-substrate attachments. The goal of the present studies was to determine whether establishment of stable contacts correlates with cessation of endothelial proliferation during corneal development in vivo. METHODS: Corneas from neonatal (days 1, 3, 7, 10, 13, 14, 17, 21, 28, and 42) and adult rats were used for immunolocalization of the following: bromodeoxyuridine (BrdU), an S-phase marker; p27kip1 and p21cip1, G1-phase inhibitors; connexin-43 and ZO-1, proteins associated with gap and tight junctions, respectively; Na+/K+-ATPase and beta3-integrin, markers of plasma membrane polarity; and fibronectin and collagen type IV, constituents of Descemet's membrane. Nuclei staining positively for BrdU were counted to determine the relative number of S-phase cells at various times after birth. Marker protein expression and localization were determined by conventional fluorescence microscopy and by confocal microscopy. RESULTS: The number of endothelial cells staining positively for BrdU gradually decreased between postnatal days 1 and 13. After postnatal day 13, positive BrdU staining was no longer detectable. During the first postnatal week, cells stained positively for the G1-phase inhibitor p27kip1 but not for p21cip1. Connexin-43 achieved its mature location by postnatal day 1. ZO-1, Na+/K+-ATPase, beta3-integrin, fibronectin, and collagen type IV achieved their mature localization patterns between postnatal days 14 and 21. CONCLUSIONS: In neonatal rat, corneal endothelial cells are still entering the cell cycle at birth, but cell cycle entry gradually decreases, so that by postnatal day 13 cells are no longer entering the S-phase. The G1-phase inhibitor p27kip1, but not p21cip1, may help mediate this inhibition. Stable cell-cell and cell-substrate contacts gradually form, and monolayer maturation is complete between postnatal days 14 and 21. The results lead to the hypothesis that, in developing rat cornea in vivo, the establishment of stable cell-cell and cell-substrate contacts initiates a cascade of events, mediated by p27kip1, which induces mitotic inhibition in the endothelial monolayer.     

Mitomycin and the human corneal endothelium

OBJECTIVE: To investigate the ultrastructural and physiologic effects of exposure of the human corneal endothelium to mitomycin at concentrations of 20 micrograms/mL and 200 micrograms/mL using electron microscopy and in vitro specular perfusion techniques. METHODS: Four pairs of corneas (with one cornea of each pair receiving balanced salt solution [BSS Plus, Alcon Laboratories, Fort Worth, Tex] and the other receiving BSS Plus with 20 micrograms/mL of mitomycin) suitable for transplantation, except for extremes of age or systemic disease, underwent perfusion with corneal thickness measured serially every 15 minutes followed by fixation for electron microscopy. Mean corneal swelling rate was calculated for all four experiments, and the control group that received BSS Plus was compared with the group that received mitomycin using a paired t test. Electron micrographs were examined in a masked fashion. Similar studies were performed using two pairs of corneas that received 200 micrograms/mL of mitomycin. RESULTS: The mean swelling rate for corneas perfused with 20 micrograms/mL of mitomycin (-4.1 microns/h) was not significantly different from that seen in tissue perfused with BSS Plus (-4.2 microns/h). No consistent ultrastructural changes could be attributed to exposure to 20 micrograms/mL of mitomycin. Perfusions of mitomycin at 200 micrograms/mL resulted in prompt corneal swelling with marked ultrastructural alterations compared with tissue perfused with BSS Plus. CONCLUSION: Human corneal endothelium may be exposed to undiluted (200 to 500 micrograms/mL) mitomycin with inadvertent entry into the anterior chamber during dissection of the scleral flap bed in trabeculectomy followed by application of mitomycin. This will result in prompt destruction of the endothelium. Exposure to 20 micrograms/mL of mitomycin, a level exceeding the concentration that may be present in the aqueous humor after its proper application, appears nontoxic in this system.     

Examination and Photography of donor corneal endothelium

In a method for the routine examination of the endothelium of donor corneas before transplantation, the donor endothelial cells are examined and photographed as they undergo preservation or culture in liquid media. A specular microscope with a modified objective lens is used (working distance, approximately 9 mm). The donor corneas are kept in a special storage container that allows a direct view of the endothelium from outside the container. The endothelium of any cornea preserved in a liquid medium may thus be examined and photographed at high magnification without opening the storage container.     

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.     

Viscoelastic adherence to corneal endothelium following phacoemulsification

OBJECTIVE: The study examined the prevalence and correlates of criminal victimization and the relationship between victimization and client outcomes for homeless clients with mental illness. METHODS: Subjects were clients in community treatment programs participating in the Access to Community Care and Effective Services and Supports (ACCESS) program of the Center for Mental Health Services. Data were obtained through interviews conducted at program entry and at three and 12 months after entry with ACCESS clients in 18 sites during the first year of program operation (N = 1,839). Self-reports of victimization during the past two months as well as data on sociodemographic, health, and social adjustment indicators were obtained at each time point. Multiple regression was used to determine both the correlates of victimization among this population and the effect of recent victimization on client outcomes three and 12 months after program entry. RESULTS: Forty-four percent of the clients were the victims of at least one crime during the two months before entering the program. Women were significantly more likely than men to have been victimized. Multivariate analysis showed that the more severe the client's psychotic symptoms, alcohol abuse, and criminal history, the more likely he or she was to have been victimized. Recent victimization had a significant impact on client outcomes in terms of increased homelessness and decreased quality of life. Victimization shortly before program entry was also the single most important predictor of victimization at both follow-up points. CONCLUSIONS: These findings suggest the critical need for service providers who work with homeless people with serious mental illness to assess the extent to which they have been victims of crime and to address issues of victimization and safety along with psychiatric and social adjustment problems.     

Adenosine stimulation of fluid transport across rabbit corneal endothelium

The rate of fluid transport across rabbit corneal endothelium has been measured with an automatic volumetric method. The present resolution of the procedure is 1-3 nanoliters, and intervals of measurement can be made as small as seconds. In the presence of glucose, oxidized glutathione (GSSG), and adenosine, the maximal rates were 6.2+/-1.0 microliter/hr cm2, and 8.2+/-0.8 microliter/hr cm2 if a large portion of the stroma was dissected away. In the presence of glucose and GSSG only, the rates were lower, namely 3.7+/-0.5 microliter/hr cm2. The rates consistently increased or decreased when adenosine was added or deleted, respectively, during given experiments. The stimulation of fluid transport by adenosine was in the order of 40-50%. The results raise the possibility that this transport mechanism might be subject to metabolic control.     

Detection of neurone-specific enolase in long-term cultures of human corneal endothelium

BACKGROUND: Human corneal endothelial cells cultivated in monolayer culture for protracted periods undergo morphological dedifferentiation, whereby they assume a more fibroblast-like appearance. These cultures may also become overgrown with contaminating stromal fibroblasts and/or with keratocytes, when non-selective media are employed, thus rendering identification of actual endothelial cells difficult on a strictly morphological basis. METHODS: The endothelium of the human cornea stains for neurone-specific enolase (NSE) in situ, and we therefore wished to study the expression of this marker in primary and long-term monolayer cultures of these cells. Ten such cultures were established, six being stained for NSE at the primary and first-passage stage, the other four for 6, 8, 10 and 12 months. The NSE-staining pattern manifested in co-cultures of corneal endothelium and fibroblasts or keratocytes (first to fifth passage cultures) was also investigated, and co-cultures established from each of the latter two cell types served as controls. RESULTS: In monolayers of corneal endothelium which had retained their cobblestone-like morphology, NSE could be demonstrated even after more than 20 passages, which amounted to 1 year in culture. Dedifferentiated or degenerating endothelial cells stained poorly and inhomogeneously. Control cultures of fibroblasts or keratocytes were consistently NSE-negative, and when each of these cell types was co-cultured separately with corneal endothelium, only the latter expressed the marker protein. CONCLUSION: Since antibodies against NSE are commercially available, practical use may be made of this marker protein for confirming corneal endothelial status in long-term cultures.     

Specular microscopic observations of the corneal endothelium in the normal rabbit

Endothelial specular microscopy and pachometry were performed on both eyes of 14 young adult New Zealand white rabbits with clinically normal eyes. Endothelial cells of the central corneas formed a mosaic-like pattern of homogenous hexagonal cells with a mean diameter of 20.6 +/- 1.0 micron sd. The mean number of cells per mm was 2998 +/- 326 sd and the mean corneal thickness was 0.38 +/- 0.02 mm sd.     

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.     

Changes of the corneal endothelium after ultraviolet-B exposure in previously photokeratectomized eyes

The photorefractive effect of excimer lasers is based on an interaction between the 193-nm ultraviolet-C laser beam and the stromal chromophore molecules. Recently, in some patients an increase of subepithelial haze and a regression of refractive effect has been observed following suntanning (UV-B exposure). The aim of the study was to find out the possible endothelial damage caused by photoablation with increasing depth and the effect of subsequent UV-B exposure on previously photokeratactomized eyes. Altogether 12 chinchilla rabbits were treated. Four animals received a -5.0 D PRK; four animals a -15.0 D PRK and four animals a -30.0 D PRK treatment. The endothelial average number, size and variation were determined two weeks post-PRK. Three weeks following PRK, a half of the animals received a 1 J/cm2 ultraviolet-B radiation in a constant dermatological UV-chamber. The endothelial morphology was measured the same way with automated specular microscopy two weeks after UV-B irradiation. After PRK treatment there was no statistically demonstrable change in endothelial morphology. On the other hand, after UV-B radiation all eyes showed a decrease in endothelial number and an increase in size and variation. The ratio of hexagonality decreased, and endothelial rosette formation appeared. The early morphological changes resembled the physiological aging changes. Conditions (deep stromal photoablation, cumulative effect of suntanning or solarium treatments) may exaggerate the physiological aging processes leading to subsequent pleomorphism, polymegatism and cell loss. This may accelerate corneal dysfunction.     

Keratocyte loss after corneal deepithelialization in primates and rabbits

PURPOSE: To evaluate the response of stromal keratocytes to central corneal deepithelialization. METHODS: Rabbits and monkeys underwent unilateral mechanical deepithelialization with a blunt instrument and were killed at intervals ranging from 15 minutes to 24 hours after surgery. Two rabbits underwent unilateral deepithelialization under a fluid bath containing corneal preservation medium. Two rabbits were treated unilaterally with corneal preservation medium topically applied every 15 minutes for 16 hours after epithelial removal. Four rabbits underwent linear keratotomy immediately after deepithelialization of the cornea or on normal unoperated corneas and were killed 1 day (two animals) and 14 days (two animals) after surgery. RESULTS: Deepithelialization resulted in severe ultrastructural changes in keratocytes within 30 minutes after surgery. After 24 hours, the number of keratocytes in the anterior stroma underneath the deepithelialized area had decreased significantly in rabbits (P = .0001) and in monkeys (P = .0007) compared with controls. The wound healing was altered and delayed when the epithelium was not present after keratotomy. The use of storage media during and after deepithelialization minimized the early keratocyte changes and appeared to stimulate reepithelialization. CONCLUSIONS: Removal of corneal epithelium causes loss of superficial stromal keratocytes in rabbits and monkeys. Keratocyte death may results from osmotic changes that alter the corneal wound healing response.     

Use of of the high frequency capsulotomy instrument in cataract surgery--effect of coagulation on corneal endothelium

Intumescent or hypermature cataracts make a safe capsulorhexis impossible. High frequency capsulotomy represents a satisfying solution for this problem. Primary goal of the present study was to investigate a possible damage to the corneal endothelium by this method. MATERIALS AND METHODS: 55 patients with an uncomplicated senile cataract were enclosed into a prospective randomized study undergoing cataract surgery with capsulorhexis or with high frequency capsulotomy. Corneal endothelium was examined preoperatively as well as postoperatively at several intervals. RESULTS: Concerning loss of endothelial cells and parameters of polymegatism and pleomorphism there were no statistically significant differences between both groups. CONCLUSION: The diathermy during high frequency capsulotomy does not show any clinically relevant negative effects on the corneal endothelium within cataract surgery.     

Excimer laser effects on human corneal endothelium. Modulation by serum factor(s)

OBJECTIVE: To determine the possibility of endothelial cell damage after excimer laser ablation. METHODS: Endothelial cell densities and morphology of human corneas after photoablations or mechanical keratectomy were compared with those of the untreated mates after 1 week of culture with or without serum. RESULTS: Corneas cultured in serum-free medium after ablation to a depth of 150 microns showed endothelial cell densities reduced to 60% of untreated, mate corneas; ultrastructural analysis showed endothelial cell damage not seen in untreated mates. Corneas ablated to the same depth and cultured in serum-enriched medium showed no endothelial cell density loss, nor did corneas cultured in serum-free medium after an ablation to a depth of 50 microns or mechanical keratectomies averaging 95 microns. CONCLUSIONS: Endothelial cell loss in deep laser resections may be prevented by factor(s) in fetal bovine serum. The apparent lack of cell loss in clinical studies may be related to the protective action of similar factors in aqueous humor.     

Relationship between fluid transport and in situ inhibition of Na(+)-K+ adenosine triphosphatase in corneal endothelium

PURPOSE: To examine the relationship between the activity of the sodium pump of the corneal endothelium and corneal thickness. It was postulated that because inhibition pressure of the stroma decreases as thickness increases, a partially inhibited sodium pump would result in a new steady-state thickness of the cornea when reduced rates of fluid influx and efflux were equal. Measurements of physiologic behavior and biochemical activity were to be made in the same tissue and thus establish the relationship directly. METHODS: Rabbit corneas were superfused with a bicarbonate Ringer solution containing different concentrations of ouabain. Exposure to ouabain was either continuous for 4 hours or for an initial 10 minutes followed by ouabain-free superfusion. Thickness was measured, and, after superfusion, endothelium was removed from the corneas, sonicated, and assayed for Na(+)-K+ adenosine triphosphatase (ATPase) activity without further addition of ouabain to the assay medium. Thickness was also measured during superfusion with suboptimal concentrations of Na+ or HCO3- and with brefeldin A, an inhibitor of protein trafficking. RESULTS: Continuous exposure to ouabain caused corneas to swell, but no new steady-state thickness was reached. At low concentrations, swelling rates increased with time, as did the extent of inhibition of the Na(+)-K+ ATPase. With only a 10-minute exposure to ouabain, swelling rates with 10(-4) M to 10(-5) M decreased with the duration of ouabain-free superfusion. Similar swelling curves were obtained by reductions in Na+ or HCO3- concentrations in the superfusion medium, indicating that partial inhibition of the endothelial fluid transport processes, whether via the Na(+)-K+ ATPase or by suboptimal ionic conditions, led toward a new equilibrium thickness of the cornea. However, when superfusion was continued for more than 4 hours, the corneas exposed for 10 minutes to 3 x 10(-5) M or lower-concentration ouabain showed increasing Na(+)-K+ ATPase activity and began to thin, indicating a recovery of fluid transport capability. This recovery was blocked by addition of brefeldin A during the ouabain-free superfusion. CONCLUSIONS: Inhibition of Na(+)-K+ ATPase by low concentrations of ouabain increases with time. Temporary exposure to ouabain causes swelling at rates that decline with time as ouabain dissociates from enzyme sites. This dissociation, together with the turnover of Na(+)-K+ ATPase in the plasma membrane, can lead to recovery of normal thickness in ouabain-exposed corneas. Twenty percent of Na(+)-K+ ATPase in the endothelium is estimated to be intracellular, and about 20% of the activity can be inhibited without inducing swelling.