Leif R. Nelson

Ten-year postoperative results of penetrating keratoplasty.

OBJECTIVE To investigate the changes in central corneal endothelial cells and corneal thickness in transplanted corneas from 5 to 10 years after grafting. This study also aimed to investigate the development of glaucoma, graft rejection, and graft failure during the first 10 postoperative years. DESIGN/PARTICIPANTS Longitudinal cohort study of 500 consecutive penetrating keratoplasties by 1 surgeon. Patients were asked to return for follow-up examinations at 2 months and at 1, 3, 5, and 10 years after grafting. The authors excluded eyes regrafted during the study and the fellow eyes of bilateral cases, leaving 394 grafts in 394 patients for analysis. INTERVENTION Penetrating keratoplasty was performed. MAIN OUTCOME MEASURES Using specular microscopy, the authors measured endothelial cell density, coefficient of variation of cell area, percentage of hexagonal cells, and corneal thickness. The authors performed clinical examinations to determine graft rejection or failure and the development of glaucoma. RESULTS By 10 years postkeratoplasty, 80 of the 394 patients had died and 68 grafts had failed. Of the remaining 246 patients, 119 (48%) returned for their 10-year examinations. For the 72 patients who returned for all of the scheduled postoperative visits and had no rejection episodes, reoperations, or failure, endothelial cell loss from preoperative donor levels at 10 years was 67 +/- 18% (mean +/- standard deviation), endothelial cell density was 958 +/- 471 cells/mm2, coefficient of variation was 0.32 +/- 0.11, hexagonal cells were 56 +/- 12%, and corneal thickness was 0.58 +/- 0.05 mm. The 5- to 10-year changes for all these values were significant (P < or = 0.004). The mean rate of late endothelial cell loss from 5 to 10 years postkeratoplasty was 4.2% per year. Eyes that were aphakic after grafting had the lowest endothelial cell loss (57 +/- 24%) and the lowest interval cell loss from 5 to 10 years postkeratoplasty (4 +/- 19%). Eyes that were phakic had the highest endothelial cell loss (73 +/- 8%) and 5- to 10-year-interval cell loss (17 +/- 31%). Eyes with posterior chamber lenses had a greater endothelial cell loss (71 +/- 9%) than did eyes with anterior chamber lenses (51 +/- 25%, P = 0.03). The 10-year cumulative risk of glaucoma, rejection, or failure was 21%, 21%, and 22%, respectively. Late endothelial failure became the major cause for graft failure, accounting for 9 of the 11 failures after 5 postoperative years. CONCLUSIONS From 5 to 10 years after penetrating keratoplasty, the annual rate of endothelial cell loss was seven times the normal rate. The endothelial cell loss, pleomorphism, polymegethism, and corneal thickness increased significantly during this time, indicating continued endothelial instability and dysfunction, resulting in an increasing rate of late endothelial failure.

Corneal Endothelîum Five Years After Transplantation

We asked the recipients of 500 consecutive corneal transplants to return for examination and endothelial photography at two months and at one, three, and five years postoperatively. Thirty-six regrafts and 70 fellow eyes of bilateral cases were excluded, leaving 394 eyes for analysis. We also recorded episodes of graft rejection and failure. In 129 grafts in patients who returned at each postoperative interval and had no rejection episodes, the mean endothelial cell density continued to decrease 7.8% per year from three years to five years after keratoplasty, compared with approximately 0.5% per year in unoperated-on normal corneas. The mean cell loss compared with the preoperative examination was 58.9% five years after keratoplasty. The percentage of hexagonal cells did not return to preoperative levels by five years after keratoplasty, suggesting that the endothelium continued to be unstable. The mean corneal thickness increased significantly with time. The Kaplan-Meier rates of rejection episodes and failure were 19% and 17%, respectively, five years after keratoplasty. Eyes with posterior chamber lens implants lost more endothelial cells by five years after keratoplasty than did eyes with open-looped anterior chamber lens implants. Low endothelial cell densities were statistically significantly associated with increased corneal thickness and with an increased risk of subsequent failure. The central endothelial cells of successful corneal transplants five years after keratoplasty form an unstable monolayer with continued accelerated loss of cells and abnormal cellular morphologic features. This process results in fewer endothelial cells remaining on the central graft with an associated increase in stromal swelling and graft failure.

Continued Endothelial Cell Loss Ten Years after Lens Implantation

PURPOSE To investigate the effects of cataract extraction and lens implantation on the central corneal endothelium 10 years after surgery. METHODS The authors conducted a prospective study of 253 consecutive eyes that underwent cataract extraction with or without lens implantation by one surgeon from 1976 to 1982. Three types of lens implant were used during this period. The protocol included ophthalmic examinations and specular microscopy on all eyes preoperatively, and 2 months and 1, 3, 5, and 10 years postoperatively. RESULTS The 10-year analysis was conducted on 67 (26%) of the 253 total eyes. The remaining patients died (86 eyes [34%]), were unable to return 10 years later (93 eyes [37%]), or had secondary implants (5 eyes [2%]) or penetrating keratoplasty (2 eyes [1%]). There were no statistically significant differences among the median 10-year endothelial cell losses of 36% in 17 control cataract extractions without lens implantation (15 extracapsular and 2 intracapsular), 40% in 15 medallion iris suture implants, 32% in 28 transiridectomy clip implants, and 32% in 7 posterior chamber implants. The median exponential rate of chronic cell loss from 1 to 10 years after surgery was 2.5% per year, which did not differ significantly among the three implant groups or between the implants (2.4% per year) and controls (2.7% per year). The chronic cell loss rate was significantly higher (7.2% per year) in six eyes with cornea guttata, which was the only preoperative endothelial morphologic feature that was significantly associated with the chronic cell loss rate. CONCLUSIONS Ten years after cataract extraction, eyes continued to lose endothelial cells from the central cornea at a rate of 2.5% per year, 2.5 to 8.0 times the rate in healthy unoperated eyes. The rate was not affected significantly by the presence of the three types of lens implants that the authors used. Postoperative eyes with cornea guttata continued to lose cells at more than twice this rate. Preoperative specular microscopy did not provide additional information helpful in predicting postoperative endothelial status or outcome.

Comparison of recording systems and analysis methods in specular microscopy

PURPOSE To compare corneal endothelial cell images from contact and automated noncontact specular microscopes and to compare endothelial image analysis by the Konan Robo Center Method and the Bio Optics Bambi Corners Method. METHODS Twenty-six normal corneas of 13 subjects and 41 penetrating keratoplasties (PKs) of 38 patients were photographed with a Keeler-Konan contact specular microscope and a Konan Noncon Robo automated noncontact specular microscope. (i) After measuring and calibrating the magnification of each instrument, we digitized the cellular apices and analyzed the images from both instruments by using the Corners Method modified to accept x and y calibrations. (ii) Using the internal calibration marks of the Konan Noncon Robo specular microscope for calibration of magnification (as required for the Center Method), we evaluated identical cells on images from this microscope by both the Center Method and the Corners Method. (iii) We evaluated the reproducibility of both methods by repeating measurements on the same image. RESULTS (i) When the images were properly calibrated for magnification by using an external scale, endothelial cell density (ECD) of normal corneas was 2,703 +/- 354 (mean +/- SD) cells/mm2 by contact and 2,685 +/- 357 cells/mm2 by noncontact techniques (p = 0.51). ECD of PK corneas was 1,767 +/- 773 cells/mm2 by contact and 1,807 +/- 775 cells/mm2 by noncontact techniques (p = 0.31). (ii) When images from the Konan Noncon Robo specular microscope were calibrated for magnification on the internal marks, the measured ECD from the same noncontact photographs was 6% less (p < 0.001). ECD was then 2,519 +/- 294 cells/mm2 (means +/- SD) by the Center Method and 2,523 +/- 305 cells/mm2 by the Corners Method (p = 0.55) in normal corneas and 1,715 +/- 748 cells/mm2 by the Center Method and 1,731 +/- 763 cells/mm2 by the Corners Method (p = 0.04) in PK corneas. (iii) The coefficient of variation of repeated measurements on the same normal image was 0.0025 for the Centers Method and 0.0099 for the Corners Method. CONCLUSIONS (i) Images from the automated noncontact specular microscope may be used interchangeably with those from the contact specular microscope to measure ECD, but only if both are properly calibrated by measuring an external scale. (ii) As a method of analysis, the Center Method is equivalent to the Corners Method in normal corneas, but the proprietary internal calibration of the Center Method, which is required for its use, yields ECDs approximately 6% less than when an external scale is used for distance calibration. (iii) Cell density measurements by both the Center Method and the Corners Method were reproducible within 1%.

Comparison of Chen Medium and Optisol-GS for human corneal preservation at 4°C: Results of transplantation

Purpose. To compare results after transplantation of donor corneas stored in Chen Medium (containing &bgr;-hydroxybutyrate without sodium bicarbonate or chondroitin sulfate) to corneas stored in Optisol-GS medium (containing sodium bicarbonate and 2.5% chondroitin sulfate). Methods. We performed 32 consecutive penetrating keratoplasties with donor corneas stored at 4°C in either Chen Medium or Optisol-GS by random assignment. Corneal thickness measurements were made at 1 day, 1 week, 3 weeks, 2 months, and 1 year postkeratoplasty. Specular microscopic images of the donor endothelium were obtained at the beginning of storage and 2 months and 1 year postkeratoplasty. The percentage of intact epithelium 1 day after keratoplasty and the graft epithelialization time were estimated by the surgeons. Donor rim cultures were performed. Results. No statistically significant differences in corneal thickness or endothelial cell loss between the corneas stored in the two media were found at any time, although differences of less than 12% cell loss or 0.09-mm thickness at 2 months or less than 25% cell loss or 0.10-mm thickness at 1 year could not be excluded with 90% certainty in this small series. The mean percentages of intact graft epithelium on day 1, 64% for Chen Medium and 65% for Optisol-GS, were not significantly different. Endothelial cell density 2 months postkeratoplasty was significantly decreased for corneas stored in both media. Endothelial cell loss at 2 months was directly correlated with storage time in both media. Conclusions. After keratoplasty, no statistically significant differences in corneal thickness, epithelial survival, and endothelial cell loss were found between corneas stored in Chen Medium and Optisol-GS. Endothelial cell loss at 2 months was significantly correlated with storage time in both media.

In vitro comparison of Chen medium and Optisol-GS medium for human corneal storage.

Purpose. To compare paired human corneas after storage at 4°C in Chen medium (CM) and Optisol-GS medium (OM) for 7, 10, 14, and 21 days. Methods. One cornea of each pair from nine human donors was randomly stored in either CM or OM, with its mate cornea stored in the other medium. Three pairs of corneas were stored for 7 days and two pairs each were stored for 10, 14, and 21 days at 4°C. Baseline corneal thickness measurements and endothelial photographs were obtained with a specular microscope. Corneal thickness measurements were also taken on days 7, 10, 14, and 21 of storage. At the end of storage, the corneas were warmed 2 hours before endothelial photographs were taken and were then placed in fixative. A corneal endothelial analysis system was used to compare changes in endothelial size and shape after storage. After fixation, the corneal endothelium was examined by scanning electron microscopy (SEM), and TdT-dUTP terminal nick-end labeling (TUNEL) assays with 4`6-diamidino-2-phenylindole (DAPI) counterstaining were performed on tissue sections of each cornea. A laser scanning confocal microscope and an automated digital analysis system were used to detect the presence of TUNEL-positive apoptotic cells in each cell layer and to determine keratocyte densities. Results. Mean corneal thickness at 0, 7, 10, 14, 21 days of storage was 0.69 ± 0.05 mm, 0.69 ± 0.06 mm, 0.73 ± 0.08 mm, 0.87 ± 0.04 mm, and 0.87 ± 0.03 mm, respectively, for CM and 0.65 ± 0.06 mm, 0.59 ± 0.07 mm, 0.63 ± 0.03 mm, 0.60 ± 0.03 mm, and 0.69 ± 0.02 mm, respectively, for OM (p < 0.0001). The mean decrease in endothelial cell density at the end of the 7-, 10-, and 14-day storage periods was 11 ± 10% for the CM corneas and 5 ± 5% for the OM corneas (p = 0.18). SEM showed an intact endothelial monolayer in all corneas. The mean percentages of TUNEL-positive cells in epithelium, stroma, and endothelium of CM-stored corneas were 4 ± 4%, 2 ± 3%, and 0.1 ± 0.3%, respectively, and did not differ from the OM-stored corneal values of 4 ± 3%, 2 ± 4%, and 0.9 ± 1.5%. The percentage of TUNEL-positive cells did not increase with storage time. Keratocyte density was 368 ± 130 cells/mm2for CM-stored corneas and 447 ± 96 cells/mm2for OM-stored corneas (p = 0.13). Conclusions. Corneas stored in CM were thicker during storage than those stored in OM. The two storage media did not differ with respect to endothelial cell loss during storage or to the percentage of TUNEL-positive cells or keratocyte density at the end of the storage period.

Transplantation of cryopreserved human corneas in a xenograft model

An ideal model to test methods of corneal storage for transplantation would simulate the environment of the grafted human cornea and predict the success of clinical corneal transplants (human to human). In this study, we tested such a model, the corneal xenograft (human to cat). Nine pairs of human corneas were transplanted into both eyes of nine recipient cats. One cornea of each pair was cryopreserved at -196 degrees C in 2.5 M dimethyl sulfoxide while the other was stored in preservative medium at 4 degrees C (control) for 6 +/- 2 (mean +/- SD) days before transplantation. One week after transplantation, the cats were euthanized and the eyes were examined. Three of the grafts (all cryopreserved) were clinical failures and showed no survival of donor corneal endothelial cells on scanning electron microscopy. The remaining six pairs of grafts were examined with a specular microscope and showed endothelial cell losses of 48 +/- 16% in cryopreserved and 8 +/- 16% in control corneas (p < 0.05). This survival is similar to survival in an earlier corneal perfusion model. The nine cryopreserved grafts were thicker than the control grafts, had fewer surviving keratocytes in the central stroma, and had more apoptotic central keratocytes (TUNEL assay). This failure rate in cryopreserved corneas clearly shows that this technique of cryopreservation was not adequate for clinical use. The corneal xenograft model can be used to study cellular survival and apoptosis in vivo after preservation as well as to test new methods of corneal preservation before initiating clinical trials.

Morphologic Assessment of Corneal Endothelium by Specular Microscopy in Evaluation of Donor Corneas for Transplantation

Our purpose was to evaluate the role of specular microscopy in the assessment of donor corneas for transplantation. We conducted retrospective analysis of specular microscopic evaluations of 1,000 consecutive donor corneas processed at Mayo Clinic Eye Bank from 1986 to 1993. Thirty-four of the 1,000 corneas were excluded from transplantation use on the basis of specular microscopic examination. Twenty-four corneas were excluded because of the presence of dark spots on the endothelium that did not clear with time. Large endothelial cells were found in six corneas on inspection, with a mean cell density of 1,160 cells/mm2 (795–1,597 cells/mm2). The remaining four excluded corneas showed evidence of endothelial trauma. Of 966 corneas not excluded, 520 (mean cell density 2,632 cells/mm2, range 1,621–4,590 cells/ mm2) were transplanted at the Mayo Clinic, and the rest were distributed for transplantation elsewhere, when possible. Six of the corneas transplanted at the Mayo Clinic (1.2%) failed primarily. There were no significant differences in the preoperative characteristics of the donor corneas between the donor failures and the clear grafts. Specular microscopic examination excluded 3.4% of donor corneas on the basis of unsatisfactory endothelium. Despite examination of the endothelium, six of 520 transplanted corneas (1.2%) suffered primary graft failure. Morphologic assessment of donor corneal endothelium by specular microscopy probably lessens, but does not eliminate, the risk of primary donor failure.

Lysosomal enzymes in corneal storage media and corneal graft outcome.

Purpose The purpose of this study was to relate lysosomal enzyme activities in corneal storage media to the outcome of the transplanted corneas. Methods Corneal storage media from 358 transplanted corneas were frozen at −70°C and kept for enzyme analysis. Corneas were stored in K-Sol (28), CSM (35), Dexsol (80), Index medium (five), Optisol (158), and Optisol GS (52). Activities of α-D-mannosidase, β-glucuronidase, α-glucosidase, and N-acetyl-β-glucosaminidase were assayed fluorometrically. Mayo Clinic records were examined for donor information, including cause of death and 2-month graft follow-up data. Results For all corneas, there was a low but significant correlation between activities of each enzyme and storage time (r-s = 0.13–0.35; p = 0.02–0.0001), and donor age (rs = −0.14 to −0.23; p = 0.009–0.0001). There was no significant correlation of enzyme activity with 2-month endothelial cell density, structure, cell loss, or corneal thickness. Enzyme activities for four primary donor failures and six grafts with <65% 2-month endothelial cell loss were not significantly different from those for the rest of the transplanted corneas. Enzyme activities were higher for corneas from donors with renal failure but not from those with diabetes mellitus. There was no significant difference in graft outcome for different cause-of-death groups. Conclusions The activities of lysosomal enzymes released into corneal storage media are not useful as predictors of graft outcome.

Specular microscopy before and after enucleation of live donor eyes

PURPOSE To compare the results of specular microscopic examination of corneal endothelium before and after enucleation of eyes from live donors. METHODS Endothelial cell density (ECD), coefficient of variation of cell area (CV), and percent hexagonal cells were compared for 34 cornea donors before enucleation of their eyes and after excision of the corneoscleral rims and placement in preservative media. RESULTS There was no statistically significant difference in ECD, CV, or percent six-sided cells after enucleation. The pre-enucleation and post-enucleation ECD measurements were significantly correlated (rs = .85, P < .0001). Mean percentage change in ECD was -0.7% +/- 6.0%. CONCLUSIONS There was no significant difference in ECD, CV, or percent six-sided cells between measurements taken from the epithelial side in vivo and those taken of the same corneas from the endothelial side in vitro after enucleation and corneoscleral rim excision. These findings suggest that it is reasonable to compare postkeratoplasty clinical measurements with those of the donor corneas taken in the eye bank.