Harry J. Menegay

Effect of incision width on graft survival and endothelial cell loss after Descemet stripping automated endothelial keratoplasty.

Purpose: To assess the effect of incision width (5.0 and 3.2 mm) on graft survival and endothelial cell loss 6 months and 1 year after Descemet stripping automated endothelial keratoplasty (DSAEK). Methods: One hundred sixty-seven subjects with endothelial decompensation from a moderate-risk condition (principally Fuchs dystrophy or pseudophakic corneal edema) underwent DSAEK by 2 experienced surgeons. The donor was folded over and inserted with single-point fixation forceps. This retrospective analysis assessed graft survival, complications, and endothelial cell loss, which was calculated from baseline donor and 6-month and 1-year postoperative central endothelial images evaluated by an independent specular microscopy reading center. Results: No primary graft failures occurred in either group. One-year graft survival rates were comparable (98% vs 97%) in the 5.0- and 3.2-mm groups, respectively (P = 1.0). Complications included graft dislocation, graft rejection episodes, and elevated intraocular pressure and occurred at similar rates in both groups (P ≥ 0.28). Pupillary block glaucoma did not occur in either group. Mean baseline donor endothelial cell density did not differ: 2782 cells per square millimeter in the 5.0-mm (n = 64) and 2784 cells per square millimeter in the 3.2-mm (n = 103) groups. Percent endothelial cell loss was 27% ± 20% (n = 55) versus 40% ± 22% (n = 71; 6 months) and 31% ± 19% (n = 45) versus 44% ± 22% (n = 62; 12 months) in the 5.0- and 3.2-mm incision groups, respectively (both P < 0.001). Conclusions: One year after DSAEK, overall graft success was comparable for the 2 groups; however, the 5.0-mm incision width resulted in substantially lower endothelial cell loss at 6 and 12 months.

Endothelial Morphometric Measures to Predict Endothelial Graft Failure After Penetrating Keratoplasty

IMPORTANCE Endothelial morphometric measures have potential value in predicting graft failure after penetrating keratoplasty. OBJECTIVE To determine whether preoperative and/or postoperative central morphometric measures (endothelial cell density [ECD], coefficient of variation [CV], and percentage of hexagonality [HEX]) and their postoperative changes are predictive of graft failure caused by endothelial decompensation after penetrating keratoplasty to treat a moderate-risk condition, principally Fuchs dystrophy or pseudophakic corneal edema. DESIGN In a subset of Cornea Donor Study participants with graft failure, a central reading center determined preoperative and postoperative ECD, CV, and HEX from available central endothelial specular images. SETTING Cornea Image Analysis Reading Center of the Specular Microscopy Ancillary Study. PARTICIPANTS Eighteen patients with graft failure due to endothelial decompensation and 54 individuals matched for most donor and recipient measures at baseline whose grafts did not fail. MAIN OUTCOME MEASURE Change in ECD, CV, and HEX values. RESULTS Preoperative ECD was not associated with graft failure (P = .43); however, a lower ECD at 6 months was predictive of subsequent failure (P = .004). Coefficient of variation at 6 months was not associated with graft failure in univariate (P = .91) or multivariate (P = .79) analyses. We found a suggestive trend of higher graft failure with lower HEX values at 6 months (P = .02) but not at the established statistical significance (P < .01). The most recent CV or HEX values, as time-dependent variables, were not associated with graft failure (P = .26 and P = .81, respectively). Endothelial cell density values decreased during follow-up, whereas CV and HEX appear to fluctuate without an apparent trend. CONCLUSIONS AND RELEVANCE Endothelial cell density at 6 months after penetrating keratoplasty is predictive of graft failure, whereas CV and HEX appear to fluctuate postoperatively, possibly indicating an unstable endothelial population in clear and failing grafts. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT00006411.

Effect of the K+/H+ ionophore nigericin on response of A549 cells to photodynamic therapy and Tert-butylhydroperoxide

The K+/H+ ionophore nigericin dramatically increases killing of V79 cells and A549 cells by photodynamic therapy (PDT) sensitized by chloroaluminum phthalocyanine. Previous studies suggested that the interaction between PDT and nigericin is related to the ability of this ionophore to reduce intracellular pH (pHi). The present study was undertaken to test the possibility that nigericin, by lowering pHi, inhibits reductive detoxification of PDT-produced peroxides by enzymes of the glutathione (GSH) redox cycle and the pentose cycle. To test this possibility we examined the effects of nigericin on the toxicity and metabolism of a model peroxide, tert-butylhydroperoxide (tert-BOOH), in A549 cells, a cell line in which the GSH redox cycle is known to be the principal pathway for reduction and detoxification of tert-BOOH. We found that nigericin equilibrates pHi of A549 cells with extracellular pH (pHe) in a time-dependent manner. It increases the toxicity of tert-BOOH toward A549 cells, inhibits loss of tert-BOOH from the buffer overlying the cells, and reduces the rate of 14CO2 release from radiolabelled glucose, which is a measure of pentose cycle activity. These effects are significantly greater at pHe 6.40 than at 7.40. Monensin, a Na+/H+ ionophore which does not reduce pHi, does not enhance the toxicity of tert-BOOH and has only a minimal effect on tert-BOOH reduction. These data suggest that nigericin-induced inhibition of peroxide detoxification is at least a plausible mechanism by which the ionophore might interact with PDT.

Inhibition of recovery from potentially lethal radiation damage in A549 cells by the K+/H+ ionophore nigericin.

A549 cells held for 4 hr in Hanks balanced salt solution, after 10 Gy irradiation, exhibit potentially lethal damage recovery (PLDR) which is dependent on extracellular pH (pHe). Recovery factors of 2.2 to 3.5 are observed when pHe is 6.40 to 7.30, but recovery factors of less than 1.0 are found when pHe is reduced to 6.20 or 6.00. The K+/H+ ionophore nigericin, when added to cells post-irradiation, inhibits PLDR in a pHe-dependent manner; it is increasingly more effective as pHe is reduced from 6.80 to 6.40. The presence of nigericin thus causes inhibition of PLDR at pHes that normally promote recovery. The drug does not affect radiation response of A549 cells when present only during irradiation. Effects of low pHe buffer, with and without nigericin, on intracellular pH (pHi) and on ATP levels were examined in an effort to elucidate the mechanisms for inhibition of PLDR and enhancement of radiation response. Incubation of cells in pHe 6.00 buffer results in a slight decrease in pHi and does not induce a drop in ATP levels. In contrast, post-irradiation incubation of cells in pHe 6.40 buffer containing 2 microM nigericin causes an immediate and dramatic decrease in pHi, and a gradual loss of ATP to 30% of control levels by 4 hr. The data obtained so far suggest that a very slight lowering of pHi may influence post-irradiation holding recovery, and that the mechanisms by which pHe 6.00 buffer alone, or pHe 6.40 buffer containing nigericin, affect holding recovery are different.

Interaction of phthalocyanine photodynamic treatment with ionophores and lysosomotrophic agents

Phthalocyanines are receiving increasing attention as second-generation sensitizers for photodynamic therapy (PDT). This paper discusses some of the investigations into the mechanism of the phototoxic responses of phthalocyanine-sensitized PDT exploiting the interaction of PDT with other metabolic modulators. Among the agents which interact strongly with PDT is the K+/H+ ionophore nigericin. Under the conditions studied with chloroaluminum phthalocyanine (AlPcCl), the Na+/H+ ionophore monensin, the Ca++ ionophore A23187, and the lysosomotrophic agent chloroquine, but not the K+ ionophore valinomycin, also potentiate photodynamic cell killing. None of the latter compounds interact with PDT as strongly as does nigericin. Both nigericin and monensin partially inhibit cellular respiration; however, KCN, which inhibits respiration completely, is less effective in potentiating PDT damage than is nigericin. Nigericin treatment alone does not deplete glutathione; however, the GSH level decreases after treatment of cells with PDT and nigericin. The potentiation of the PDT response is much greater at an extracellular pH (pHe) of 6.70 than at pHe 7.30. When nigericin is present at pHe 6.70, the intracellular pH (pHi) is equilibrated with pHe. None of the other ionophores tested was able to cause the acidification of the intracellular milieu as did nigericin. The evidence to date suggests that the lowering of pHi is an important component of the mechanism by which nigericin potentiates PDT.

Donor, Recipient, and Operative Factors Associated With Increased Endothelial Cell Loss in the Cornea Preservation Time Study

Importance Determining factors associated with endothelial cell loss after Descemet stripping automated endothelial keratoplasty (DSAEK) could improve long-term graft survival. Objective To evaluate the associations of donor, recipient, and operative factors with endothelial cell density (ECD) 3 years after DSAEK in the Cornea Preservation Time Study. Design, Setting, and Participants This cohort study was a secondary analysis of data collected in a multicenter, double-masked, randomized clinical trial. Forty US clinical sites with 70 surgeons participated, with donor corneas provided by 23 US eye banks. Individuals undergoing DSAEK for Fuchs dystrophy or pseudophakic/aphakic corneal edema were included. Interventions The DSAEK procedure, with random assignment of a donor cornea with a preservation time of 0 to 7 days or 8 to 14 days. Main Outcomes and Measures Endothelial cell density at 3 years as determined by a reading center from eye bank and clinical specular or confocal central endothelial images. Results The study included 1090 participants (median age, 70 years) with 1330 affected eyes (240 bilateral cases [22.0%]), who underwent DSAEK for Fuchs dystrophy (1255 eyes [94.4%]) or pseudophakic/aphakic corneal edema (PACE) (75 eyes [5.6%]). Of these, 801 eyes (60.2%) belonged to women and 1207 (90.8%) to white individuals. A total of 749 participants (913 eyes; 164 [21.9%] bilateral cases) had functioning grafts with acceptable endothelial images preoperatively and at 3 years postoperatively and were included in this analysis. Factors associated with a lower ECD at 3 years (estimated effect with 99% CI) in the final multivariable model included donors with diabetes (−103 [−196 to −9] cells/mm2), lower screening ECD (−234 [−331 to −137] per 500 cells/mm2), recipient diagnosis of PACE (−257 [−483 to −31] in cells/mm2), and operative complications (−324 [−516 to −133] in cells/mm2). Endothelial cell loss (ECL) from a preoperative measurement to a 3-year postoperative measurement was 47% (99% CI, 42%-52%) for participants receiving tissue from donors with diabetes vs 43% (99% CI, 39%-48%) without diabetes; it was 53% (99% CI, 44%-62%) for participants diagnosed with PACE vs 44% (99% CI, 39%-49%) for those diagnosed with Fuchs dystrophy, and 55% (99% CI, 48%-63%) in participants who experienced operative complications vs 44% (99% CI, 39%-48%) in those who did not. No other donor, recipient, or operative factors were significantly associated with 3-year ECD. Conclusions and Relevance Donor diabetes, lower screening ECD, a PACE diagnosis in the recipient, and operative complications were associated with lower ECD at 3 years after DSAEK surgery and may be associated with long-term graft success. While causation cannot be inferred, further studies on the association of donor diabetes and PACE in recipients with lower 3-year ECD warrant further study.

Donor endothelial cell density measurements do not change immediately after DMEK preparation

Purpose: To evaluate a single eye banks measurement of endothelial cell density (ECD) of Descemet membrane endothelial keratoplasty (DMEK) grafts before and after preparation using 2 separate counting methods. Methods: A series of 60 donor tissues were prepared for DMEK surgery. One to 4 specular images of the central endothelium were taken both before and after preparation, and ECDs were evaluated for a total of 345 unique images. Images were then masked and provided to the Cornea Image Analysis Reading Center (CIARC) for independent analysis. Results: Before preparation, average eye bank-determined ECD with the center method was 2678 ± 259 cells/mm2 and was 2599 ± 280 cells/mm2 CIARC-determined by the variable frame method (P < 0.001, n = 176). After preparation, eye bank-determined ECD was 2719 ± 265 cells/mm2 and CIARC-determined ECD was 2615 ± 344 cells/mm2 (P < 0.001, n = 169). The difference in ECD before and after DMEK preparation was not found to be statistically significant when evaluated using either analysis method (P = 0.19; P = 0.64) before and after preparation, respectively. Conclusions: Although the absolute ECD value may differ by the analysis method statistically, pre- and post-DMEK preparation ECDs did not significantly change by either analysis method. Other methods such as vital staining to assess tissue damage after preparation in conjunction with specular microscopy are suggested.