Cynthia R. Reed

Eye bank survey of surgeons using precut donor tissue for descemet stripping automated endothelial keratoplasty.

PURPOSE To assess surgeon satisfaction with precut corneal tissue from 1 eye bank for Descemet stripping automated endothelial keratoplasty (DSAEK). Surgical techniques and predictors of procedural success were also examined. METHODS A 19-question survey was completed by 53 surgeons around the United States for 197 DSAEK cases using prepared corneal allograft tissue from the Iowa Lions Eye Bank. Surgeries were performed between April 1 and December 31, 2006; surveys were completed retrospectively within a few weeks of surgery. RESULTS Tissue was found to be acceptable in 98% of DSAEK cases reported. Difficulties with precut tissue (eg, lack of anterior cap adherence to the posterior lamella, not visible or decentered central dot, anterior edge undermining) were reported in approximately 10% of cases. A rebubbling procedure was performed in 23% of cases for donor dislocations. The donor lenticule adhered, with resulting corneal deturgescence, in 86% of cases. Surgeons declared a successful procedure in 92% of cases. Of the 14 unsuccessful cases, donor tissue quality was the underlying etiology in only 1 case. Procedural success rates were related to surgeon experience (P = 0.002), lenticule adherence after only 1 anterior chamber air bubble (P = 0.005), no small perforations to release fluid (P = 0.005), and the presence of corneal deturgescence (P = 0.002). CONCLUSIONS The use of precut tissue for DSAEK is not associated with increased risk of complications related to tissue preparation. With standardization of precutting donor tissue, safety of DSAEK surgery may be improved while increasing surgeon efficiency.Background:Syphilis outbreaks among men who have sex with men (MSM) in the United States have raised concerns about increased HIV transmission in this population. We sought to estimate HIV incidence among men diagnosed with primary or secondary (P&S) syphilis in sexually transmitted disease (STD) clinics in Atlanta, San Francisco, and Los Angeles. Methods:We analyzed deidentified sociodemographic information from routine syphilis surveillance databases and matching remnant sera from consecutive male patients with P&S syphilis who were tested for syphilis at 3 public health laboratories during January 2004 through January 2006. Deidentified sera positive for Treponema pallidum by particle agglutination were screened for HIV-1 antibodies by enzyme immunoassay (EIA). Specimens that were confirmed HIV-positive by Western blot analysis were then tested for recent HIV infection using the less sensitive (LS) HIV-1 Vironostika EIA and BED HIV-specific IgG/total IgG assay. Results:Of 357 men with P&S syphilis (98 in Atlanta, 151 in San Francisco, and 108 in Los Angeles), 32% had primary syphilis and 85% were MSM (12% no MSM risk and 3% no information). The median age was 36 years; 40% were white, 31% black, 20% Hispanic, and 8% other. Among men with P&S syphilis, 160 (45%) were HIV-positive, of whom 8 were classified as having acquired recent HIV infection by the LS-Vironostika EIA (all confirmed by BED) and had no history of antiretroviral use or HIV-positive results >6 months earlier. Seven of the 8 men with recent HIV infection were MSM. The estimated HIV incidence was 9.5% per year (95% confidence interval [CI]: 2.9 to 16.0) among all men and 10.5% per year (95% CI: 2.7 to 18.3) among MSM. Conclusions:We found high HIV incidence among a high-risk population of US men diagnosed with P&S syphilis in STD clinics in Atlanta, San Francisco, and Los Angeles. Intensive integrated HIV/STD prevention programs are needed for this population.

Mitochondrial and Morphologic Alterations in Native Human Corneal Endothelial Cells Associated With Diabetes Mellitus

Purpose To characterize changes in the energy-producing metabolic activity and morphologic ultrastructure of corneal endothelial cells associated with diabetes mellitus. Methods Transplant suitable corneoscleral tissue was obtained from donors aged 50 to 75 years. We assayed 3-mm punches of endothelium-Descemet membrane for mitochondrial respiration and glycolysis activity using extracellular flux analysis of oxygen and pH, respectively. Transmission electron microscopy was used to assess qualitative and quantitative ultrastructural changes in corneal endothelial cells and associated Descemet membrane. For purposes of analysis, samples were divided into four groups based on a medical history of diabetes regardless of type: (1) nondiabetic, (2) noninsulin-dependent diabetic, (3) insulin-dependent diabetic, and (4) insulin-dependent diabetic with specified complications due to diabetes (advanced diabetic). Results In total, 229 corneas from 159 donors were analyzed. Insulin-dependent diabetic samples with complications due to diabetes displayed the lowest spare respiratory values compared to all other groups (P ≤ 0.002). The remaining mitochondrial respiration and glycolysis metrics did not differ significantly among groups. Compared to nondiabetic controls, the endothelium from advanced diabetic samples had alterations in mitochondrial morphology, pronounced Golgi bodies associated with abundant vesicles, accumulation of lysosomal bodies/autophagosomes, and focal production of abnormal long-spacing collagen. Conclusions Extracellular flux analysis suggests that corneal endothelial cells of donors with advanced diabetes have impaired mitochondrial function. Metabolic findings are supported by observed differences in mitochondrial morphology of advanced diabetic samples but not controls. Additional studies are needed to determine the precise mechanism(s) by which mitochondria become impaired in diabetic corneal endothelial cells.

Assessing the Impact of Diabetes Mellitus on Donor Corneal Endothelial Cell Density

Purpose: To quantify changes in endothelial cell density (ECD) of donor corneal tissue in relation to the presence or absence of a medical history of diabetes mellitus diagnosis, treatment, and complications. Methods: A retrospective review was performed for all corneas collected at Iowa Lions Eye Bank between January 2012 and December 2015. For purposes of analysis, donor corneas were divided into 4 groups: nondiabetic, non–insulin-dependent diabetic, insulin-dependent diabetic without medical complications due to diabetes, and insulin-dependent diabetic with medical complications due to diabetes. ECD values (obtained through specular microscopy) and transplant suitability for endothelial transplantation (determined by the standard protocol of the eye bank) were compared among groups using linear mixed model analysis. Results: In total, 4185 corneas from 2112 donors were included for analysis. Insulin-dependent diabetic samples with medical complications due to diabetes (N = 231 from 119 donors) showed lower ECD values compared with nondiabetic samples (−102 cells/mm2, P = 0.049) and non–insulin-dependent diabetic samples (−117 cells/mm2, P = 0.031). ECD values did not differ significantly among the remaining groups. The likelihood of suitability for endothelial transplantation did not differ among all 4 groups. Conclusions: Corneas from donors with insulin-dependent diabetes mellitus and medical complications resulting from the disease have lower mean ECD values compared with other donors. However, our analysis suggests that these corneas are equally likely to be included in the donor pool for corneal transplantation. Additional studies are needed to determine the mechanism(s) contributing to cell loss in donors with advanced diabetes and to assess associated endothelial cell functional impairment.

Proteomic analysis of corneal endothelial cell-descemet membrane tissues reveals influence of insulin dependence and disease severity in type 2 diabetes mellitus

The objective of this study was to characterize the proteome of the corneal endothelial cell layer and its basement membrane (Descemet membrane) in humans with various severities of type II diabetes mellitus compared to controls, and identify differentially expressed proteins across a range of diabetic disease severities that may influence corneal endothelial cell health. Endothelium-Descemet membrane complex tissues were peeled from transplant suitable donor corneas. Protein fractions were isolated from each sample and subjected to multidimensional liquid chromatography and tandem mass spectrometry. Peptide spectra were matched to the human proteome, assigned gene ontology, and grouped into protein signaling pathways unique to each of the disease states. We identified an average of 12,472 unique proteins in each of the endothelium-Descemet membrane complex tissue samples. There were 2,409 differentially expressed protein isoforms that included previously known risk factors for type II diabetes mellitus related to metabolic processes, oxidative stress, and inflammation. Gene ontology analysis demonstrated that diabetes progression has many protein footprints related to metabolic processes, binding, and catalysis. The most represented pathways involved in diabetes progression included mitochondrial dysfunction, cell-cell junction structure, and protein synthesis regulation. This proteomic dataset identifies novel corneal endothelial cell and Descemet membrane protein expression in various stages of diabetic disease. These findings give insight into the mechanisms involved in diabetes progression relevant to the corneal endothelium and its basement membrane, prioritize new pathways for therapeutic targeting, and provide insight into potential biomarkers for determining the health of this tissue.

Assessing the Effects of Ripasudil, a Novel Rho Kinase Inhibitor, on Human Corneal Endothelial Cell Health

PURPOSE To determine how the Rho kinase inhibitor, ripasudil, affects metabolic function and cell viability in donor human corneal endothelial cells (HCECs). METHODS Endothelial cell-Descemet membrane (EDM) tissues were treated with 10 μM ripasudil and assayed for mitochondrial and glycolytic activity using extracellular flux analysis and then compared to untreated controls. In addition, EDM tissues with a 24-h ripasudil treatment and control tissues were exposed to 1 μM staurosporine to induce apoptosis and then analyzed for cell viability using apoptosis and necrosis assays. RESULTS Mitochondrial respiration metrics, specifically maximal respiration (P = 0.758) and spare respiratory capacity (P = 0.777), did not differ among the 1-h ripasudil treatment, 24-h treatment, and untreated tissues. Glycolytic activity assays showed an increase in glycolytic capacity at 1 h compared to the 24-h exposure group (P = 0.049) and controls (P = 0.009). Following exposure to staurosporine, the percentage of apoptotic HCECs was lower (P = 0.009) in ripasudil-treated tissues (2.473%, standard error of the mean [SEM] 0.477%) compared to untreated controls (3.349%, SEM 0.566%). In contrast, the percentage of necrotic HCECs decreased but did not differ statistically (P = 0.158) between ripasudil-treated (3.789%, SEM 0.487%) and untreated (4.567%, SEM 0.571%) tissues. CONCLUSIONS Exposures to ripasudil did not result in any detectable reduction in metabolic function for HCECs in an ex vivo donor tissue model, and an increase in glycolytic activity at the 1-h time point was detected. In addition, HCECs treated with ripasudil gained a protective effect against induced apoptosis, suggesting that ripasudil may help improve the integrity of the corneal endothelium.

A Simple and Reliable Technique to Orient Donor Corneal Tissue Using the Radial Width of the Surgical Limbus.

Purpose: To develop a method based on identification of the widest region of the surgical limbus that can yield quick and accurate orientation of excised human donor corneas. Methods: Corneoscleral tissue from donors 49 to 75 years old was marked at the temporal sclera at the time of recovery. Digital images obtained from 20 corneas stored in viewing chambers, retroilluminated and viewed from the endothelial side, were used to quantify the per-degree radial width of the surgical limbus, defined as the distance from the scleral spur to clear cornea. To evaluate differences in radial width among regions, measurements were compared with the intracorneal mean limbal width, and a per-degree z-score was calculated by averaging among corneas. Using images of corneas with the temporal mark masked and the sidedness known, 6 observers were subjected to a blinded trial of 10 corneas to determine the central point of the widest limbal region of each cornea. Results: Compared with the intracorneal mean, the mean radial width of the surgical limbus was greatest in the superior quadrant, and the difference compared with the inferior, nasal, and temporal quadrants was significant (P < 0.0001). The superior region was identified with 100% accuracy in blinded trials. The average absolute difference between the predicted and actual central point of the superior limbus was 9.75 ± 0.30 degrees. Conclusions: The radial width of the surgical limbus is greatest in the superior region of the cornea and can be used as a diagnostic feature to orient donor corneal tissue.

Eye Banking: What the Eye Bank Can Do for You Now

Corneal surgeons cannot operate without their local eye banks, and eye banks would not exist without corneal surgeons. They both have the same purpose: to provide safe and effective tissue for patients in order to restore vision and eliminate corneal blindness. In fact, with recent innovations in corneal tissue preparation, corneal surgery begins in the eye bank. In this chapter, we review the history of eye banking, medical standards and federal oversight, serological testing, and the risk of disease transmission addition, techniques of harvesting and processing corneas for full-thickness penetrating keratoplasty, DSEK, DMEK, and femtosecond laser-assisted keratoplasty are reviewed. The role of donor corneas in keratolimbal allograft transplantation (KLAL) for severe ocular surface disease (OSD) is discussed, as well as the role of the eye bank in the production of autologous serum eye drops. New innovations for increasing tissue donation and corneal tissue preparation are discussed. As new techniques are developed requiring transplantation of thinner and thinner layers of the cornea eye banks continue to develop and perfect their techniques, to learn more about which donors work best for which surgeries, and to partner with physicians in the provision of precut tissues.