Evaluation of the endothelium of human donor corneas by induced dilation of intercellular spaces and trypan blue

The endothelium of 30 pairs of human cadaver corneas was stained by trypan blue and the intercellular spaces were visualized by induced dilation prior to corneal culture. Trypan blue staining and induced dilation of intercellular spaces by 0.9% and 0.45% NaCl were found to be atraumatic. Only a fraction of damaged cells were stained by trypan blue. Endothelial cell losses in culture did not correlate with the number of trypan-blue stained cells, the post-mortem time, or donor age.     

Poloxamer 188 enhances endothelial cell survival in bovine corneas in cold storage

PURPOSE: To improve corneal endothelial cell survival during cold preservation by the addition of a compound that enhances cell membrane repair. METHODS: Cell survival was assessed using intracellular accumulation of the fluorescent molecule calcein as a marker for endothelial cell viability in bovine corneas stored at 4 degrees C in Optisol-GS. The effect on endothelial cell survival of artificially lowering the surface tension of the cell membrane was also assayed by the addition of a surfactant, Poloxamer 188 (PLURONIC F68), which has been shown to reseal damaged cell membranes, even under conditions where metabolism is inhibited. RESULTS: Endothelial cell survival in corneas stored at 4 degrees C in Optisol-GS was increased by the addition of 1 mg/mL Poloxamer 188. The level of dead or missing corneal endothelial cells was less than 1% for corneas stored for 6, 9, or 12 days in Optisol-GS with 1 mg/mL Poloxamer 188 at 4 degrees C. In contrast, dead or missing corneal endothelial cell percentages were increased to approximately 4% after 9 days and approximately 10% after 12 days in storage in Optisol-GS without Poloxamer 188 at 4 degrees C. CONCLUSION: The surfactant Poloxamer 188 enhances endothelial cell survival in cold-stored bovine corneas.     

Evaluation of repeatability for the automatic estimation of endothelial cell density in donor corneas

BACKGROUND/AIMS: The repeatability of an automatic system for estimation of endothelial cell density (ECD) from microscopy images in donor corneas was assessed. METHODS: A computer program for the automatic ECD estimation from frequency analysis was previously presented. An evaluation of its repeatability was performed on a data set containing 200 corneas by assessing the accuracy and precision of automatic versus manual values. For each cornea, 2-21 images (1536 total) at 100x for automatic ECD and one image at 200x for manual ECD were available. RESULTS: Accuracy of automatic ECDs was -45 (SD 99) cells/mm(2) (-2% (4%)). Precision of repeated automatic ECDs on the same cornea was 62 (30) cells/mm(2) (3% (1%)). The algorithm was also capable of identifying all non-processable images. CONCLUSION: The proposed automatic technique proved to be reliable for its routine use in a typical eye bank setting like the one considered here.     

Endothelial cell density in porcine corneas after exposure to hypotonic solutions

Purpose To evaluate exposure to sucrose solution (1.8%) and hypotonic balanced salt solution (BSS) for its effects on endothelial cell density of porcine corneas.Methods Two groups of central discs from pig corneas were organ-cultured for 24 h. Twelve corneas per group were exposed to sucrose solution (1.8%) or hypotonic BSS for 4 min each. The paired corneal discs were not treated and served as controls. After further organ culture with and without dextran for 48 h, corneal endothelium was stained with alizarin red and examined by light microscopy. The endothelial cell densities were determined manually on three central images.Results The endothelial cell density differed significantly between corneas exposed to sucrose and the control corneas (3982±382 cells/mm² and 4360±331 cells/mm² respectively, and 3876±364 cells/mm² versus 4374±168 cells/mm² respectively with 6% dextran). In contrast, the endothelial cell density did not differ significantly between corneas exposed to hypotonic BSS and the control corneas (4374±296 cells/mm² and 4317±193 cells/mm² respectively, and 4348±151 cells/mm² versus 4426±175 cells/mm², respectively with 6% dextran).Conclusions Exposure to 1.8% sucrose for 4 min induces a significant endothelial cell loss of 10% on average, whereas exposure to hypotonic BSS did not significantly influence the endothelial cell density.Presented in part at the annual meeting of the Deutsche Opthalmologische Gesellschaft (DOG), Berlin, 2005.     

A new system for the automatic estimation of endothelial cell density in donor corneas

AIMS: The problem of automatic estimation of endothelial cell density from microscopy images in donor corneas was addressed. METHODS: The spatial frequencies contained in digital endothelium images are extracted with a two dimension discrete Fourier transform (DFT) technique. A circular band in the DFT of the images is shown to contain the frequency information related to the cell density. An algorithm for reliably recovering this spatial frequency information and for extracting from it an estimate of endothelial cell density has been developed and implemented in a computer program. An evaluation was performed on a data set containing 100 donor corneas, by comparing automatic values with manual counts performed by three eye bank experts on two images for each cornea. RESULTS: The mean difference of automatic densities v manual ones was 14 cells/mm(2) (0.9%), with a standard deviation of 119 cells/mm(2) (5.1%) and mean absolute difference of 92 cells/mm(2) (3.9%). The ANOVA based overall inter-rater reliability was 0.935. The algorithm was also capable of identifying all non-processable images. Running times were in the order of 1-2 seconds per image. CONCLUSION: A new algorithm was developed for the fully automatic estimation of endothelial cell density. The results of a clinical evaluation on 100 corneas suggest that it is capable of reliably estimating endothelium cell density in donor corneas.     

Corneal endothelial cell density after trabeculoplasty

Central corneal endothelial photographs were taken with a specular microscope. The cell density was calculated with a computerized image analyzer. No difference between the treated and control eyes could be found. The laser energy level delivered in trabeculoplasty seems to be too small to induce any permanent changes in the endothelial cells.     

Factors affecting bovine corneal endothelial cell density in vitro.

AIMS: To examine factors influencing the density and contact inhibition of bovine corneal endothelial cells cultured in vitro. METHODS: Cell counts were performed on bovine corneal endothelial cells cultured for various times in the presence of 10% fetal calf serum, with or without varying concentrations of growth factors, 5% dextran T-500, or 2% chondroitin sulphate, at 32 degrees C or 37 degrees C, and after treatment with beta galactosidase. RESULTS: Both basic fibroblast growth factor (FGFb) and retinal crude extract (RCE), but neither epidermal growth factor (EGF) nor acidic fibroblast growth factor (FGFa), increased endothelial cell density in vitro (p < 0.05). Continuous exposure to RCE resulted in a higher cell density than did a 24 hour pulse (p < 0.01), and higher cell densities were achieved at 37 degrees C than at 32 degrees C (p < 0.0001). In the absence of RCE, dextran T-500 increased cell density modestly (p < 0.05); in the presence of RCE, the addition of dextran T-500 had no effect on final cell density, whereas chondroitin sulphate significantly decreased final cell density (p < 0.01). In the absence of exogenous growth factors, beta galactosidase treatment resulted in a 50% increase in final cell density compared with controls (p < 0.0001). CONCLUSIONS: Bovine corneal endothelial cell growth can be augmented under conditions different from those used in corneal preservation systems. The final cell density in a confluent monolayer can be increased by treatment with beta galactosidase, suggesting that corneal endothelial cells may be contact inhibited through a beta galactosidase sensitive receptor system.     

Gap junctional intercellular communication in bovine corneal endothelial cells

Gap junctions and/or paracrine mediators, such as ATP, mediate intercellular communication (IC) in non-excitable cells. This study investigates the contribution of gap junctions toward IC during propagation of Ca(2+) waves in cultured bovine corneal endothelial cells (BCEC) elicited by applying a point mechanical stimulus to a single cell in a confluent monolayer. Changes in [Ca(2+)](i) were visualized using the fluorescent dye Fluo-4. The area reached by the Ca(2+) wave, called the active area (AA), was determined as a measure of efficacy of IC. RT-PCR and Western blotting showed expression of Cx43, a major form of connexin, in BCEC. In scrape-loading (using lucifer yellow) and fluorescence recovery after photobleaching (FRAP; using carboxyfluorescein) protocols, significant dye transfer of the hydrophilic dyes was evident indicating functional gap junctional IC (GJIC) in BCEC. Gap27 (300 microM), a connexin mimetic peptide that blocks gap junctions formed by Cx43, reduced the fluorescence recovery in FRAP experiments by 19%. Gap27 also reduced the active area of the Ca(2+) wave induced by point mechanical stimulation from 73,689 microm(2) to 26,936 microm(2), implying that GJIC contribution to the spread of the wave is at least approximately 63%. Inhibitors of ATP-mediated paracrine IC (PIC), such as a combination of apyrase VI and apyrase VII (5U/ml each; exogenous ATPases), suramin (200 microM; P2Y antagonist), or Gap26 (300 microM; blocker of Cx43 hemichannels) reduced the active area by 91%, 67%, and 55%, respectively. Therefore, estimating the contribution of GJIC from the residual active area after PIC inhibition appears to suggest that GJIC contributes no more than approximately 9% towards the active area of the Ca(2+) wave. Gap27 did not affect the enhancement in active area induced by ARL-67156 (200 microM, ectonucleotidase inhibitor), ATP release induced by point mechanical stimulation, and zero [Ca(2+)](o)-induced lucifer yellow uptake, indicating that the peptide has no influence on PIC. Exposure to Gap27 in the presence of PIC inhibitors led to a significant further inhibition of the Ca(2+) wave. The finding that the residual active area after inhibition of PIC by apyrases was much smaller than the reduction of the active area by Gap27, provides evidence for interaction between GJIC and PIC. These findings together suggest that functional gap junctions are present in BCEC, that both GJIC and PIC contribute significantly to IC, and that the two pathways interact.     

ATP-dependent paracrine intercellular communication in cultured bovine corneal endothelial cells

PURPOSE: Intercellular communication (IC) in nonexcitable cells is mediated through gap junctions and/or through the release of paracrine mediators. This study was conducted to investigate adenosine-5' triphosphate (ATP)-dependent paracrine IC in the propagation of Ca2+ waves in confluent monolayers of cultured bovine corneal endothelial cells (BCECs). METHODS: A Ca2+ wave was induced by point mechanical stimulation (PMS) of a single cell by indentation with a glass micropipette (approximately 1 microm tip) for <1 second. Dynamic changes in [Ca2+]i in the mechanically stimulated (MS) cell and in the neighboring (NB) cells were visualized with a confocal microscope, using a fluorescent dye. Normalized fluorescence (NF), calculated as the ratio of the average fluorescence of a cell to the average under resting conditions, was used as a measure of [Ca2+]i. Expression of P2Y receptors and ecto-adenosine triphosphatases (ATPases) was investigated by RT-PCR. ATP release in response to PMS was measured by luciferin-luciferase (LL) bioluminescence. RESULTS: BCECs subjected to PMS showed a transient [Ca2+]i increase. Under control conditions, the maximum NF in the MS cell occurred within 600 ms, and the fluorescence returned to baseline within 170 seconds. NB cells also presented a [Ca2+]i increase with a transient characterized by decreasing maximum NF and increasing latency as a function of the distance from the MS cell. These transients propagated as an intercellular Ca2+ wave to a distance of five or six NB cells away from the MS cell, covering areas (called active areas, AAs) up to 77,000 +/- 3,200 microm2 (N=21). The percentage of responsive cells (defined as cells showing maximum NF >1.1) decreased with increasing distance from the MS cell. The Ca2+ wave crossed cell-free lanes. Pretreatment of cells with the nonselective purinergic receptor antagonist suramin (200 microM), exogenous apyrases, which break down nucleotides (10 U/mL), or the PLC inhibitor U-73122 (10 microM) reduced the wave propagation, whereas the ecto-ATPase inhibitor ARL-67156 (100 microM) significantly enhanced it. ATP-dependent LL bioluminescence increased after PMS. RT-PCR showed mRNAs for P2Y1 and P2Y2 receptors and ecto-ATPases in BCECs. CONCLUSIONS: PMS of BCECs induces release of ATP and a concomitant intercellular Ca2+ wave, even in the absence of direct cell-cell contacts. The AA of the wave is modulated by agents that affect P2Y receptor activity. Thus, PMS-induced intercellular Ca2+ wave propagation in BCECs involves ATP-dependent paracrine IC.     

Increased endothelial cell loss after transplantation of corneas preserved by a modified organ-culture technique

Forty-seven donor corneas were preserved in McCarey-Kaufman (M-K) medium at 4 degrees C for 1 day, then in organ culture at 34 degrees C for approximately 1 month, then in M-K medium at 4 degrees C for an additional two days before transplantation. The central donor endothelium was examined by specular microscopy before and after organ culture and 2 months after keratoplasty. No significant change in central endothelial cell density occurred during organ culture. The 47 transplants were compared with 47 grafts preserved only in M-K medium at 4 degrees C for approximately 36 hours. All transplants were performed by the same surgeon over the same period, and the two groups contained similar types of surgical procedures. The organ-cultured grafts were thicker on the first post-operative day and took longer to epithelialize . Two months after keratoplasty all of the 94 grafts were clear and thin, but the mean central endothelial cell loss was 28% in the 47 organ-cultured transplants and 10% in the 47 transplants preserved only in M-K medium (P less than 0.0001). These results indicate that the endothelium of corneas preserved by organ culture at 34 degrees C and then placed in M-K medium at 4 degrees C for 2 days may be more susceptible to surgical trauma than those preserved only in M-K medium at 4 degrees C.     

The coating of bovine and rabbit corneas denuded of their endothelium with bovine corneal endothelial cells.

A method for coating corneas denuded of their endothelium has been developed. The attachment and spreading of cultured bovine corneal endothelial cells seeded upon the Descemet's membrane of corneal buttons previously denuded of their endothelium by delicately sweeping the endothelial side with a cotton swab have been analyzed. Confluent cultures of bovine corneal endothelial cells were exposed to trypsin to disrupt the cell monolayer into single cells. Increasing concentrations of endothelial cells ranging from 2·5 × 10⁴ to 3 × 10⁵ cells were then seeded on the denuded Descemet's membrane of 11 mm bovine corneal buttons. When the corneal buttons were stained with alizarin red after an incubation period of 8 hr at 37°C, the best coating was observed with 10⁵ seeded cells. In this case, no areas of denudation could be seen and the cells were clearly apposed to one another, thereby reconstituting an endothelial cell monolayer. The cultured bovine corneal endothelial cells seeded on denuded Descemet's membranes plated extremely rapidly. By 15 min, 80% of Descemet's membrane was covered with a monolayer of endothelial cells and by 30 min all of Descemet's membrane was covered.The plating of bovine corneal endothelial cells on denuded Descemet's membrane was a direct function of the trypsin concentration to which they were first exposed. Cells first treated with 0·05% trypsin plated poorly 1 hr after being seeded on a denuded Descemet's membrane. Better plating efficiency was achieved with cells first exposed respectively to 0·025% and 0·01% trypsin. The best results were consistently obtained with cells first dissociated with 0·005% trypsin.Although serum is required in vitro for the attachment of normal cells to tissue culture dishes, it was not required for the attachment of corneal endothelial cells to the denuded Descemet's membrane. Cultured corneal endothelial cells plated equally well in the presence or absence of serum. Similar results were obtained when the cells were suspended in aqueous humor instead of in tissue culture medium.When denuded rabbit corneas were used as a substrate instead of bovine corneas, all the parameters studied for the attachment and spreading of bovine corneal endothelial cells seeded on bovine corneas (cell density, time, and medium) lead to similar conclusions with respect to the interactions between corneal endothelial cells and rabbit Descemet's membrane.     

Corneal endothelial cell density after an attack of acute glaucoma

The corneal endothelial cells of 25 patients with unilateral acute glaucoma were photographed with a clinical specular microscope. Photography was performed as soon as (average 6-12 h) the IOP had been lowered with iv Acetazolamide and/or Mannitol and topical Pilocarpine therapy, and the corneas had become clear. Peripheral iridectomy was performed on the affected eye and prophylactically on the fellow eye in most of these patients. The follow-up endothelial photographs were taken 6-24 months postoperatively. High intraocular pressure lasting 3 days or more lowered the central endothelial cell density. But a rise in pressure lasting from only a few hours to 2 days did not affect the endothelial cell count. Operative glaucoma procedures caused a loss of central endothelial cells of approximately 4.8% in the series. There was a clear correlation between the duration of elevated pressure and the number of central corneal endothelial cells lost.     

Alterations in endothelial cell density after photorefractive keratectomy with adjuvant mitomycin

PURPOSE: To elucidate the distinct role of the intraoperative use of mitomycin C (MMC) on endothelial cell density after photorefractive keratectomy (PRK) in human eyes. DESIGN: Prospective, double-masked, randomized clinical trial. METHODS: One eye of 15 patients was treated with PRK with intraoperative use of topical 0.02% MMC (15 seconds), whereas the fellow eye was treated with Epipolis laser in situ keratomileusis (Epi-LASIK) in random order. Corneal confocal microscopy was performed in all eyes preoperatively and at one, three, six, and 12 months after the surgery. Moreover, three endothelial images were acquired in each of 15 preoperative-normal eyes to evaluate the repeatability of measuring endothelial cell density. Repeated measures analysis of variance was used to compare the temporal variations of endothelial cell density between the two techniques and the changes of endothelial cell density over time. RESULTS: The coefficient of repeatability of endothelial cell count was 148 cells/mm(2). Preoperative endothelial cell density was not significantly different between the two groups (P = .82). Moreover, the effect of the treatment on the temporal variation of endothelial cell density was insignificant (P = .83), whereas the differences between the preoperative and the postoperative endothelial cell densities reached statistical significance (P .05). CONCLUSIONS: The prophylactic intraoperative application of MMC (up to 15 seconds) after PRK does not seem to affect the endothelial cell density.     

Decreased endothelial cell survival after transplantation of corneas preserved by three modifications of corneal organ culture technique

We placed human donor corneas in M-K medium at 4 degrees C for 24 hours, cultured them in minimal essential medium at 34 degrees C for two to five weeks, and then either (1) placed the corneas in M-K medium at 4 degrees C for 48 hours before transplantation (Group 1, 47 eyes); (2) placed the corneas in M-K medium at 4 degrees C for 16 hours before transplantation (Group 2, 17 eyes); or (3) transplanted the corneas without postculture cooling to 4 degrees C (Group 3, 11 eyes). We compared the corneas preserved by organ culture with an equal number of corneas transplanted during the same period, but preserved only in M-K medium at 4 degrees C for one to four days. The central endothelial cell losses noted two months after keratoplasty were significantly greater in the organ-cultured corneas than in the M-K-preserved corneas in each of the three groups. The mean endothelial cell loss in the 11 organ-cultured corneas in Group 3 was significantly less than that in the 64 organ-cultured corneas in Groups 1 and 2. The corneas in Group 1 were also examined one year after keratoplasty, and the cell losses in the organ-cultured grafts remained significantly greater than those in the M-K-preserved grafts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Corneal endothelial cell density in iridocyclitis

The endothelial cells of both eyes of 60 unilateral iridocyclitic patients were photographed with a specular microscope to establish the possible effect of iridocyclitis on corneal endothelial cells. Chronic severe iridocyclitis with mutton-fat keratic precipitates (KP) lowered the central endothelial cell count. Five patients out of the seven with mutton-fat KPs had a distinctly lower central cell density in the affected eye. In the remaining patients, no significant difference in cell densities could be demonstrated between affected and healthy eyes. Neither the inflammatory process itself nor the round white KPs had a deleterious effect on the central corneal endothelial cell densities.     

Time analysis of corneal endothelial cell density after cataract extraction.

Serial endothelial photographs were taken preoperatively and postoperatively in 200 eyes; 111 eyes contained a Rayner iris clip lens, 54 eyes contained a Fyodorov Sputnik lens, and 35 eyes had no lens. Central endothelial cell density was changed in all instances, with counts in implanted eyes declining 25 to 30%, and in nonimplanted eyes 10 to 15%. In both instances, the decline essentially ceased at about three months. The cause of the greater decline in implanted eyes appeared to be mechanical and subsequent cell loss after the 90-day period was virtually equal for the two groups. Methods that may be used to alter the difference in cell density occurring with implantation are best analyzed by using the 90-day period data for comparison.     

Use of a serum-free medium for long-term storage of human corneas. Influence on endothelial cell density and corneal metabolism

BACKGROUND: The success of long-term corneal organ culture is limited by the progressive loss of endothelial cells during culture and the use of culture medium supplemented with fetal calf serum as a possible source of contamination with infectious agents. In this study, we investigated the suitability of a serum-free medium (Endothelial-SFM) to improve preservation conditions for human donor corneas. METHODS: Six pairs of corneas were stored in Minimum Essential Medium (MEM) supplemented with 2% fetal calf serum (FCS) for 8-14 days. One cornea of each pair was then further cultivated in Endothelial-SFM supplemented with 2% FCS or in MEM with 2% FCS, respectively. In a second series of experiments, the endothelial cell density of seven pairs of freshly isolated donor corneas was determined during cultivation in Endothelial-SFM with 2% FCS or serum-free Endothelial-SFM. RESULTS: After precultivation in conventional medium, the endothelial cell density of corneas allocated to cultivation in Endothelial-SFM was 1000-1950 cells/mm2 and that of those subsequently cultured in MEM 1200-2000 cells/mm2. At 9 weeks, cell densities of 900-1500 cells/mm2 were found after cultivation in Endothelial-SFM compared with a total cell loss in MEM. Freshly isolated corneas cultured in Endothelial-SFM with or without FCS supplementation showed a decrease of endothelial cell density of about 20% within the first 2 weeks of storage. During further cultivation cell density remained constant without statistically significant differences between the groups. Glucose consumption of the corneas was higher in Endothelial-SFM than in MEM. Corneas stored in Endothelial-SFM with 2% FCS showed a higher glucose consumption than those preserved in serum-free Endothelial-SFM. CONCLUSION: Organ culture of human donor corneas using the serum-free basal medium Endothelial-SFM is superior to conventional culture conditions because the decrease in endothelial cell density can be ameliorated, the culture period can be prolonged and the risk of transmitting infectious agents via serum can be minimised.