Simple Modification of a DSAEK Carrier Enables Graft Insertion by Injection Without Compromising Endothelial Cell Viability.

PURPOSE: We evaluate the quality and feasibility of preloading Descemet stripping automated endothelial keratoplasty (DSAEK) grafts into a modified EndoGlide Ultrathin system for graft injection. METHODS: DSAEK grafts were prepared by experienced processing technicians at 2 separate locations, loaded into a modified EndoGlide Ultrathin, and placed in storage media. Grafts processed at one location were shipped cross-country overnight to the other location and were examined on arrival for positioning within the modified EndoGlide Ultrathin. All grafts were ejected and analyzed for endothelial cell loss (ECL) with calcein acetoxymethyl staining and FIJI segmentation. A subset of grafts was measured by optical coherence tomography for graft thickness 1 hour after cut, 1 hour after loading, and 1 day after loading. RESULTS: No grafts were displaced from the modified carrier over 3 shipping events (n = 9), and all grafts (n = 18) were successfully ejected. Grafts loaded into the modified carrier and ejected exhibited no more cell loss than grafts loaded into the standard carrier and removed by pull-through (14.0% ± 2.8% vs. 12.2% ± 3.4%, respectively, P = 0.24). Carrier modification skills can be successfully transferred as grafts loaded by a processing technician new to carrier modification were within the acceptable limit of 25% ECL for transplant DSAEK grafts. Graft thickness increased significantly (P < 0.05) between the postcut and 1-hour postload measurement and the postcut and 24-hour postload measurement. CONCLUSIONS: The EndoGlide Ultrathin can be modified to enable its use for graft injection while not compromising the ability to use the pull-through method for graft delivery. Preloaded DSAEK grafts swell significantly during the 24-hour storage period, and patterns of ECL may be linked to swelling.

Prospective In Vitro Comparison of Kerasave and Optisol-GS Corneal Storage Solutions.

PURPOSE: The aim of this study was to compare the performance of Kerasave and Optisol-GS for hypothermic corneal storage for 14 days. METHODS: This study was a prospective laboratory investigation. Mate corneas were recovered into Kerasave or Optisol-GS (27 pairs) and stored at 2°C to 8°C for 14 days. Corneas were evaluated by trained eye bank technicians, and study parameters were compared between the initial and final evaluations. Endothelial cell density (ECD), hexagonality (HEX), and coefficient of variation (CV) were evaluated by specular microscopy, and central corneal thickness (CCT) was examined by optical coherence tomography after 1, 3, 7, and 14 days of storage. Corneal transparency was scored using slit lamp examination at days 1 and 14. RESULTS: Average ECD, HEX, and CV for the Kerasave (2653 ± 303 cells/mm 2 , 57 ± 4%, and 36 ± 3%) and Optisol-GS (2623 ± 306 cells/mm 2 , 57 ± 5%, and 36 ± 4%) groups were not significantly different at day 1. There was also no difference at any other study time points (all P > 0.05). ECD did not significantly change from day 1 to day 14 in either group ( P > 0.05), but a statistically significant change in HEX and CV was observed between day 1 and day 14 in both groups ( P < 0.01). Average CCT measured at day 1 for corneas stored in Kerasave was 622 ± 49 µm and those stored in Optisol-GS was 580 ± 35 µm ( P < 0.01). The difference in CCT measurements was not significantly different at day 14 (Kerasave: 674 ± 46 µm vs. Optisol-GS: 647 ± 58 µm, P > 0.05). Corneal transparency was not significantly different between the 2 groups at day 1 or day 14. CONCLUSIONS: The corneal quality and clinically relevant parameters including ECD, endothelial morphometry, and corneal transparency were not different in corneas stored in Kerasave or Optisol-GS for 14 days. The initial difference in CCT between the 2 groups decreased at day 14. These results demonstrated that Kerasave corneal storage solution preserves the corneal endothelium similarly to Optisol-GS.

Rate of Endothelial Cell Loss and Graft Survival in Descemet Membrane Endothelial Keratoplasty in Eyes Requiring a Rebubble.

PURPOSE: We aimed to compare the rate of 6-month endothelial cell loss (ECL) and 6-month graft survival in eyes that did not require a postoperative rebubble with eyes that did require a postoperative rebubble after Descemet membrane endothelial keratoplasty (DMEK) surgery. METHODS: A consecutive series of DMEK surgeries performed from September 2013 to March 2020 was retrospectively analyzed. Eyes that did not require a rebubble for graft detachment were compared with eyes with 1 rebubble and eyes with 2 or more rebubbles for 6-month ECL and graft survival. A subanalysis of the rebubble rate for different indications for transplantation was also performed. RESULTS: One thousand two hundred ninety-eight eyes were included in this study. The 6-month ECL for eyes with no rebubbles, 1 rebubble, and ≥2 rebubbles was 29.3% ± 16.2% (n = 793), 36.4% ± 18.6% (n = 97, P = 0.001), and 50.1% ± 19.6% (n = 28, P < 0.001), respectively. The 6-month graft survival rate for eyes with no rebubbles, 1 rebubble, and ≥2 rebubbles was 99.5%, 97.8% ( P = 0.035), and 81.8% ( P < 0.001), respectively. When compared to the rebubble rate for DMEK for Fuchs dystrophy (156/1165 eyes = 13.4%), the rebubble rates were statistically higher for DMEK for failed penetrating keratoplasty (28.5%, P = 0.021) and pseudophakic bullous keratopathy (28.0%, P = 0.036). CONCLUSIONS: Eyes undergoing any rebubble procedure in the postoperative period after DMEK have an increased risk of endothelial cell loss and graft failure at 6 months postoperative. DMEK in eyes for failed penetrating keratoplasty and failed DMEK had the highest rebubble rate, with the former reaching statistical significance.

Double-Scroll Formation by Fluid Column Manipulation in Preloaded DMEK Grafts Prepared From Younger and Older Donor Tissue.

PURPOSE: The purpose of this study was to determine whether manipulation of preloaded single-scroll Descemet membrane endothelial keratoplasty (DMEK) grafts within the fluid column of an injector can safely and reliably result in formation of double-scroll DMEK grafts and whether there are differential effects on younger versus older donor tissue. METHODS: Pairs of DMEK grafts prepared from older (65-80 years) and younger (48-64 years) donors were preloaded into a Straiko modified Jones tube. One member of the pair was manipulated within the fluid column to form a double-scroll graft, and the other remained unmanipulated. Outcomes measured include success rate for double-scroll formation, endothelial cell loss (ECL), and relative scroll width. RESULTS: Older donor grafts formed double scrolls with a 100% success rate. ECL of older donor manipulated grafts was statistically higher than that of unmanipulated mate grafts (17.4% ± 3.5% vs. 13.0% ± 4.2%, P = 0.03), but was still within the acceptable range for transplant. Younger donor grafts were successfully manipulated into double scrolls with a 67% success rate, and there was no difference in the ECL of manipulated and unmanipulated grafts (15.5% ± 4.4% vs. 13.0% ± 4.5%, P = 0.24). For all grafts and conformations, there was a significant relationship between relative scroll width and ECL ( P < 0.01). CONCLUSIONS: Fluid column manipulation can be used reliably to form double-scroll DMEK grafts. For younger donor grafts, manipulation yields a double scroll without increasing ECL. For older donor grafts, manipulation results in a minimal, acceptable increase in ECL. Surgeons should weigh the advantage of an easily opened graft against the risk of increased ECL when considering this technique.

Examination of a Modified Graft Preparation Technique to Induce Double-Scroll Formation and Promote the Use of Younger Descemet Membrane Endothelial Keratoplasty Donor Tissue.

PURPOSE: The purpose of this study was to determine whether controlled balanced salt solution (BSS) bursts during graft preparation can safely promote formation of a double-scrolled Descemet membrane endothelial keratoplasty (DMEK) graft in younger donor tissue. METHODS: DMEK grafts prepared from young donor tissue (average age, 55 years; range, 39-66 years) were floated in BSS to spontaneously form scrolls (N = 10 pairs). Controlled BSS bursts were used to promote double-scroll (DS) formation in 1 member of each pair. Grafts were stained, preloaded, and shipped before cell viability analysis. After appropriate training, a less experienced technician performed this technique on 10 additional corneas. Outcomes measured for both technicians include the success rate for obtaining a DS, scroll conformation after shipping, and endothelial cell loss (ECL). RESULTS: There was no difference in ECL between grafts subjected to additional manipulation compared with unmanipulated mate grafts (observer 1: 15.2% ± 3.3% vs. 15.2% ± 4.4%, P = 0.99; observer 2: 16.3% ± 2.9% vs. 15.9% ± 4.5%, P = 0.8). A technician experienced with this technique had a 90% success rate, whereas a less experienced technician had a 70% success rate. The mean ECL of the 10 grafts manipulated by the less experienced technician was not significantly different from results obtained from the experienced technician (observer 1: 18.5% ± 6.0% vs. 15.2% ± 3.3%, P = 0.15; observer 2: 18.1% ± 5.6% vs. 16.3% ± 2.9%, P = 0.34). Scrolls maintained their conformation during shipping events. CONCLUSIONS: Double-scroll graft formation using controlled BSS bursts is a reliable technique that can be performed without causing additional damage to DMEK grafts. This technique may make graft unscrolling easier and can promote the use of younger donor tissue for DMEK.

Modified procedure for Loading "Flat" DMEK Grafts Into an Injector.

PURPOSE: The aim of this study was to determine whether loading a Descemet membrane endothelial keratoplasty (DMEK) graft using a drop-in procedure results in more endothelial cell loss (ECL) than the standard suction procedure. METHODS: Pairs of donor corneas with equivalent preprocessing endothelium were prepared using the standard protocol of our eye bank. One member of each pair was loaded into an injector using the standard suction protocol. The mate graft was loaded using a drop-in protocol, in which the edge of the graft was gently grasped with a forceps, lifted to the edge of the injector, and dropped inside. Grafts were evaluated for ECL and examined for grab marks or other loading-associated damage. RESULTS: There was no difference in mean ECL of grafts prepared for DMEK using the standard protocol (20.6% ± 4.5%) compared with that of mate grafts prepared using the drop-in loading protocol (19.5% ± 4.8%, P = 0.59). There was no consistent pattern of damage in the drop-in-loaded grafts, as grab marks or other tissue damage associated with the drop-in loading protocol were not consistently identified by a trained corneal surgeon. CONCLUSIONS: ECL was not significantly different in grafts prepared using a drop-in loading procedure compared with grafts prepared using the standard suction protocol. The drop-in loading protocol may be particularly useful to surgeons who load their own grafts and eye bank processing technicians who encounter a "flat" DMEK graft that does not scroll or a loosely scrolled DMEK graft.

Primary Graft Failure, Infection, and Endothelial Cell Density in Corneal Transplants With Increased Death-to-Preservation Time.

PURPOSE: To ascertain whether death-to-preservation time (DPT) is associated with donor endothelial cell density (ECD), primary graft failure (PGF), and infection. METHODS: Donor corneas aged older than 10 years with ECD 2000 to 4500 cells/mm2 were procured between 2011 and 2018 by a single eye bank. Donor corneas were analyzed retrospectively for the main outcome measures of PGF, infection, and ECD. Means and proportions of study parameters were compared between corneas with long and short DPT, defined as greater or less than 14 hours, respectively, excluding corneas with a history of intraocular surgery or diabetes. Multivariate analyses were performed using logistic regression, adjusting for donor age at time of death, history of diabetes mellitus, and history of cataract surgery. RESULTS: Among 12,015 corneas, those with long DPT had a statistically but not clinically significant higher ECD than that of corneas with short DPT (2754 vs. 2724 cells/mm2, P < 0.01). There was no difference in PGF and infections in corneas with long versus short DPT (0.28% vs. 0.26%, P = 0.86; 0.43% vs. 0.29%, P = 0.51, respectively). CONCLUSIONS: Longer DPT is not associated with a clinically meaningful reduction in donor ECD, PGF, or infection.

Ink Retention and Endothelial Cell Viability After the Application of an Orientation Stamp Over an Air Bubble During Descemet Membrane Endothelial Keratoplasty Graft Preparation.

PURPOSE: To investigate stamp visibility and endothelial cell loss (ECL) after the application of an orientation mark to Descemet membrane endothelial keratoplasty (DMEK) grafts supported by an air bubble. METHODS: Eighteen DMEK grafts were prepared at an eye bank using a technique where an orientation mark was applied to the stromal surface of a DMEK graft that was supported by a small air bubble placed at the edge of the 2 endothelial surfaces of the graft. Grafts were evaluated at 2 and 5 days for stamp visibility and at 5 days with calcein-AM staining for ECL. Nine grafts underwent cross-country shipping, and the ECL of shipped and nonshipped grafts was compared using unpaired t test. RESULTS: All 18 DMEK grafts exhibited a single, solid, readily visible orientation mark 2 and 5 days after preparation with a mean ECL of 13.5% ± 4.9%. Shipping conditions had no effect on stain retention or ECL. CONCLUSIONS: The application of an orientation stamp to a DMEK graft over an air bubble in an eye bank setting results in a single, solid orientation mark that is readily visible within the period in which most eye bank-prepared tissue is used. This technique produces no further ECL compared with the methods where the orientation stamp is applied through a stromal window. Eye bank technicians and surgeons can be confident that this modified preparation technique results in transplant-quality DMEK grafts with the additional benefit of conserving the stromal cap for use in other anterior lamellar procedures, thereby making efficient use of donor tissue.

Younger Donor Tissue in Descemet Membrane Endothelial Keratoplasty Surgery: Clinical Outcomes.

PURPOSE: To determine whether using younger donor tissue for Descemet membrane endothelial keratoplasty (DMEK) surgery influences clinical outcomes. METHODS: Scroll tightness, unscrolling time, rebubble rate, and preoperative and 3- and 6-month postoperative endothelial cell density (ECD) and endothelial cell loss (ECL) were compared for 661 DMEK grafts prepared from younger (aged younger than 50 yrs, n = 81) and older donors (aged 50 yrs or older, n = 580) with Student t test, χ2 test, or Mann-Whitney U test. RESULTS: There was no difference in overall unscrolling time (younger donors: 3.1 ± 3.1 min, older donor: 2.9 ± 2.7 min, P = 0.503). Experienced faculty surgeons, compared with fellows, had a significantly lower unscrolling times for both younger donors (2.4 ± 2.3 vs. 4.6 ± 3.9 min, P = 0.002) and older donors (2.5 ± 2.1 vs. 3.7 ± 3.3 min, P <0.001). Rebubble rates were not statistically different between younger (12.3%) and older donors (15.0%, P = 0.527). Three-month ECD was higher in grafts from younger compared with that in those from older donors (2138 ± 442 vs. 1974 ± 470 cells/mm2, P = 0.024). Six-month ECD was similar for younger (1972 ± 509 cells/mm2) and older donors (1947 ± 460 cells/mm2, P = 0.585). There was no difference in 3- or 6-month ECL comparing younger (3-mo: 24.3% ± 13.4%; 6-mo: 31.1% ± 15.2%) with older donors (3-mo: 25.9% ± 15.5%, P = 0.489; 6-mo: 27.8% ± 15.1%, P = 0.231). CONCLUSIONS: DMEK grafts prepared from younger donors exhibited similar unscrolling times, rebubble rates, and 3- and 6-month ECL compared with older donors. Experienced surgeons might begin to accept DMEK grafts from younger donors with confidence.

Efficacy of Amphotericin B in Corneal Preservation Media After Extended Frozen Storage.

PURPOSE: To investigate the antimycotic activity of amphotericin B deoxycholate that has been previously frozen for 28 days before supplementation of Optisol-GS. METHODS: Triplicate Optisol-GS samples were inoculated with 10 colony-forming units (CFU) of Candida albicans. Each set of triplicate cultures was supplemented with 2.5 µg/mL of amphotericin B that was either freshly resuspended and never frozen, frozen overnight at -20°C and thawed, or frozen at -20°C for 4 weeks and thawed. The cultures were stored at 4°C, with aliquots taken at 0, 6, 24, and 72 hours for quantification. The efficacy of each preparation of amphotericin B in reducing C. albicans growth was assessed at these time points. RESULTS: Six hours after antifungal supplementation, there was a 1.33 log10 CFU reduction with freshly resuspended amphotericin B, compared with a 1.31 log10 reduction with amphotericin B that was frozen overnight (P = 0.20) and a 1.18 log10 reduction with amphotericin B that was frozen for 4 weeks (P = 0.05). After 72 hours, there was a 2.72 log10 CFU reduction with freshly resuspended amphotericin B, a 2.64 log10 CFU reduction with amphotericin B that was frozen overnight (P = 0.45), and a 2.18 log10 CFU reduction with amphotericin B that was frozen for 4 weeks (P = 0.05). CONCLUSIONS: Previously frozen amphotericin B remains highly effective against C. albicans. Optisol-GS supplemented with 2.5 µg/mL amphotericin B that was frozen for 4 weeks at -20°C resulted in >90% CFU reduction by 6 hours and >99% reduction by 72 hours.

Donor DMEK Tissue Characteristics: Association With Rebubble Rate and 6-Month Endothelial Cell Loss.

PURPOSE: To determine whether specific donor characteristics influence postoperative rebubble rate and 6-month endothelial cell loss (ECL) in Descemet membrane endothelial keratoplasty (DMEK). METHODS: A retrospective analysis of a consecutive series of 857 DMEK surgeries using eye bank-prepared donor tissue was performed between September 2013 and April 2018. DMEK graft characteristics including donor age, preoperative endothelial cell density (ECD), preservation time, death-to-preservation time, and donor diabetes status were analyzed for correlation with rebubble rate and 6-month postoperative ECL. Subgroup analyses of donor age, preoperative ECD, preservation time, death-to-preservation time, preparation-to-surgery time, and diabetes severity were also performed. Statistically significant relationships between donor characteristics and rebubble rate or 6-month postoperative ECL were determined using Pearson correlation, one-way analysis of variance, t test, and χ analysis. RESULTS: The overall rate of rebubble after 857 surgeries performed by 7 surgeons during the study period was 12.6%. There was no significant relationship between postoperative rebubble rate and donor age, preoperative ECD, preservation time, death-to-preservation time, preparation-to-surgery time, or donor diabetes status. The subgroup analysis of these characteristics also yielded no significant relationship with rebubble rate. There was also no significant relationship between 6-month postoperative ECL and analyzed donor factors. CONCLUSIONS: Donor characteristics such as higher donor age, lower preoperative ECD (<2500), longer preservation time, and donor diabetes did not increase the rebubble rate or the 6-month ECL after DMEK. These results indicate that common surgeon preferences for donor tissues that are younger, fresher, with higher cell count, and without diabetes do not translate into superior postsurgical outcomes.

Lithium protects against anesthesia-induced developmental neuroapoptosis.

BACKGROUND: Ethanol and anesthetic drugs trigger neuroapoptosis in the developing mouse brain. Recently, it was found that ethanol-induced neuroapoptosis is preceded by suppressed phosphorylation of extracellular signal-regulated protein kinase (ERK), and lithium counteracts both the phosphorylated ERK suppressant action and ethanol-induced neuroapoptosis. The current study was undertaken to address the following questions. (1) Do ketamine and propofol mimic ethanol in suppressing ERK phosphorylation? (2) If they do, does lithium prevent this suppressant action and also prevent these anesthetic drugs from triggering neuroapoptosis? METHOD: Postnatal day 5 mice were treated with propofol, ketamine, lithium, a combination of propofol or ketamine with lithium or saline, and their brains were prepared for Western blot analysis or histology. For Western blot, cytosolic lysates of caudate putamen were analyzed for expression of phosphorylated ERK and phosphorylated serine/threonine-specific protein kinase. For histology, brains were stained immunohistochemically with antibodies to activated caspase-3, and the density of activated caspase-3 positive cells was determined. RESULTS: Ketamine and propofol suppressed phosphorylated ERK, and lithium counteracted both the phosphorylated ERK suppressant action and neuroapoptotic action of these anesthetic drugs. CONCLUSION: If further testing finds lithium to be safe for use in pediatric/obstetric medicine, administration of a single dose of lithium before anesthesia induction may be a suitable means of mitigating the risk of anesthesia-induced developmental neuroapoptosis.

Ethanol causes and lithium prevents neuroapoptosis and suppression of pERK in the infant mouse brain.

Transient exposure of immature animals during the brain growth spurt period to ethanol triggers neuroapoptosis in the developing brain. Here we report that lithium, when administered in a single, well-tolerated dose to infant mice, suppresses spontaneous neuroapoptosis that occurs naturally in the developing brain, and prevents ethanol from triggering neuroapoptosis. To explore lithium's mechanism of action, we focused on kinase signaling systems (ERK, Akt, JNK) that are believed to play a regulatory role in cell survival, and found that very rapidly after ethanol administration there is a suppression of ERK phosphorylation, and that lithium stimulates ERK phosphorylation and prevents ethanol from suppressing this phosphorylation process. Ethanol also suppressed pAKT, but lithium did not counteract this effect. We also found that ethanol activates the JNK system, but this cannot explain the neurotoxic action of ethanol, because JNK activation did not occur in the same neuronal populations that are killed by ethanol.

Subanesthetic doses of propofol induce neuroapoptosis in the infant mouse brain.

Drugs that block N-methyl-d-aspartate glutamate receptors or that promote gamma-aminobutyric acid type A inhibition trigger neuroapoptosis in the developing rodent brain. Propofol reportedly interacts with both gamma-aminobutyric acid type A and N-methyl-d-aspartate glutamate receptors, but has not been adequately evaluated for its ability to induce developmental neuroapoptosis. Here we determined that the intraperitoneal (i.p.) dose of propofol required to induce a surgical plane of anesthesia in the infant mouse is 200 mg/kg. We then administered graduated doses of propofol (25-300 mg/kg i.p.) and found that doses >or=50 mg/kg induce a significant neuroapoptosis response. We conclude that propofol induces neuroapoptosis at 1/4 the dose required for surgical anesthesia.

Treatment with a serotonin-depleting regimen of MDMA prevents conditioned place preference to sex in male rats.

Among young adults, 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy") is a popular drug of abuse, and anecdotal evidence indicates that repeated use of MDMA may result in impairments in sexual function and decreased sex drive in human users. There has been little investigation of the effects of MDMA on sexual function in rodents. In the present study, the authors determined that in male rats (Rattus novegicus) tested in a sexually naïve or a sexually experienced state, administration of a serotonin (5-HT)-depleting regimen of MDMA did not produce a change in mount, intromission, and ejaculation latency or in mount and intromission frequency compared with such latency and frequency in vehicle-treated control rats. In contrast to vehicle-treated rats, MDMA-treated rats did not form a conditioned place preference (CPP) to sex. Failure of MDMA-treated rats to form CPP to sex may be due to MDMA-induced impairments in circuits mediating sexual reward.