Canine Descemet Stripping Endothelial Keratoplasty with a Tissue Insertion Device: Technique and Long-Term Outcome.

Introduction. We describe a case of canine Descemet's stripping endothelial keratoplasty (DSEK) using an open-source canine tissue delivery device. Case Presentation. We follow the four-year outcomes of a 1.5-year-old Tibetan Terrier who presented with difficulty seeing, diffuse corneal edema, and central corneal thickness of 1400 microns in the left eye. To perform DSEK, a polycarbonate carrier and insertion device was designed for canine corneas that measure up to 15 mm in diameter. The tissue was loaded into the inserter prior to surgery with the endothelium facing inwards and the stroma facing the cartridge wall. From the cartridge, the graft was pulled into the eye using microforceps and an anterior chamber maintainer. We assessed preoperative endothelial cell count, postoperative corneal clearance, and graft adhesion. The donor was a two-year-old Airedale Terrier who died one day prior to surgery, with endothelial cell density of 3149 cells/mm2. One week after DSEK, the cornea began to clear, and pachymetry of the donor and graft total was 1410 microns. This improved to 800 microns at 4 months and continued improving in its clarity at the last postoperative visit 4 years after surgery. Discussion. We demonstrate the feasibility of conducting canine endothelial keratoplasty with a specially designed tissue delivery device and the potential of long-term corneal clearance after DSEK in canine eyes.

Viability of preloaded Descemet membrane endothelial keratoplasty grafts with 96-hour shipment.

OBJECTIVE: To assess feasibility and compare the effects of 96-hour shipment of Descemet membrane endothelial keratoplasty (DMEK) grafts as a scroll or a tri-fold on cell viability. METHODS AND ANALYSIS: DMEK grafts were prepared at the Rocky Mountain Lions Eye Bank. Twenty pre-stripped DMEK grafts, paired from 10 donors, were either tri-folded in an endothelium-in configuration using microforceps and loaded into a plastic Treyetech cartridge, or suctioned in a scrolled endothelium-out configuration into a modified Jones Tube. Grafts were shipped via FedEx to a secondary location and back for 48 hours each way, resulting in a total shipping time of 96 hours. After shipping, grafts were removed from inserters onto glass slides and unfolded using viscoelastic with endothelium facing upwards. Calcein-AM stained grafts were imaged with a fluorescent microscope and endothelial cell loss (ECL) was measured using trainable segmentation in Fiji by a masked grader. RESULTS: A total of 20 grafts were shipped for 96 hours, split between preloaded tri-folded (n=10) and preloaded scrolled (n=10) tissues. No significant difference in ECL was observed across groups after prolonged shipping (14.8% vs 13.7% ECL respectively, p=0.68). CONCLUSION: For preloaded DMEK after 96 hours, both scrolled and tri-folded tissue demonstrated clinically acceptable levels of ECL. The data suggest a wider window of time for endothelial cell viability and is promising for the prospect of international shipment of preloaded grafts.

Genome and transcriptome of a pathogenic yeast, Candida nivariensis.

We present a highly contiguous genome and transcriptome of the pathogenic yeast, Candida nivariensis. We sequenced both the DNA and RNA of this species using both the Oxford Nanopore Technologies and Illumina platforms. We assembled the genome into an 11.8 Mb draft composed of 16 contigs with an N50 of 886 Kb, including a circular mitochondrial sequence of 28 Kb. Using direct RNA nanopore sequencing and Illumina cDNA sequencing, we constructed an annotation of our new assembly, supplemented by lifting over genes from Saccharomyces cerevisiae and Candida glabrata.

Preloading Trifolded Grafts for Descemet Membrane Endothelial Keratoplasty Affects Scroll Formation.

PURPOSE: The trifolded, endothelium-in approach to Descemet membrane endothelial keratoplasty (DMEK) facilitates tissue insertion into the anterior chamber. We hypothesized that preloading the trifolded donor grafts in a cartridge for 48 hours before insertion would induce biomechanical changes that decrease their scrolling tendency compared with those loaded immediately before insertion. METHODS: Ten Descemet membrane donor grafts, peeled and cut to 8.0 mm, were prepared by a single eye bank technician. Each graft was trifolded and pulled into a DMEK cartridge and stored for 48 hours. They were then pulled with microforceps into a petri dish filled with balanced salt solution. A video was recorded of the graft becoming a scroll over a 2-minute period. Each graft, serving as its own control, was then trifolded, pulled into the cartridge, and the process repeated. Images from 1, 5, 10, 60, and 120 seconds were extracted from video recording of the procedures. Scroll width was analyzed by graders masked to group assignment. A paired t test was used to determine differences in scroll width at each time point between the 48-hour and instant trifolding conditions. RESULTS: All grafts scrolled after removal from the cartridge into balanced salt solution. We measured a significant difference at all time points 1 through 120 seconds (4.02 preloaded vs. 2.91-mm instant trifold, P = 0.035). CONCLUSIONS: Preloading DMEK grafts in a trifolded configuration for 48 hours reduces the scrolling tendency of Descemet membrane for at least 2 minutes.

Aspiration of Tri-folded, Endothelium-In Grafts for Descemet Membrane Endothelial Keratoplasty.

PURPOSE: In Descemet membrane endothelial keratoplasty (DMEK), loading and positioning of tri-folded grafts into a cartridge is generally conducted with forceps or a hook, risking graft tear or trauma. We demonstrate the feasibility of loading tri-folded grafts into a cartridge with no touch to the endothelium required beyond the tri-folding process. METHODS: A corneoscleral rim with a prestripped DMEK graft is placed into a petri dish. After the graft is tri-folded with forceps and removed from its stromal attachment, the graft is gently wicked into the tip of a saline-filled Alcon B IOL cartridge connected to IV extension tubing and a 3 cc syringe, drawn into the cartridge by positioning it adjacent to the graft tip. The remainder of the graft is aspirated with the addition of saline. The cartridge orientation is reversed for graft injection. In this retrospective analysis, we analyzed surgical videos for preparation times, and assessed postoperative visual acuity, pachymetry, and endothelial cell density. RESULTS: Thirteen cases underwent this approach. Median preparation time from stain to cartridge eye contact was 8.5 minutes, and time from graft injection to final centration and bubbling was 2.9 minutes. Corneal thickness decreased from a median of 623 microns preoperatively to 566 µm at 1 month (P = 0.038). Visual acuity improved by 1 month by a median of 0.3 logarithm of the minimum angle of resolution (logMAR) (P = 0.016). Endothelial cell density decreased by 32.4% at 1 month compared with baseline. CONCLUSIONS: Endothelium-in DMEK grafts may be loaded into a plastic cartridge using a skill set similar to aspiration of a scroll.

Comparison of Tri-folded and Scroll-based Graft Viability in Preloaded Descemet Membrane Endothelial Keratoplasty.

PURPOSE: To compare corneal endothelial damage associated with 2 techniques for preloaded Descemet membrane endothelial keratoplasty (DMEK): a tri-folded graft stored in a plastic cartridge designed for DMEK and a scrolled graft stored in a modified Jones Tube, at the time of preparation and after shipping. METHODS: DMEK grafts were prepared at the Rocky Mountain Lions Eye Bank. The grafts were either tri-folded and loaded in a plastic cartridge or scrolled and loaded into a modified Jones Tube. In each group, the grafts were then either immediately removed from the cartridges or shipped for 48 hours. The grafts were then stained with Calcein AM and imaged using a fluorescent microscope. Endothelial cell loss (ECL) was determined using trainable segmentation in Fiji by 2 graders. At each time point, rates of ECL loss were compared across the 2 groups. To explore the role of donor characteristics, a multivariable regression model was produced to account for method (tri-folding vs. scroll), donor age, donor gender, death-to-preservation time, death-to-preparation time, and shipping. RESULTS: A total of 40 grafts were prepared, processed, imaged, and analyzed. No significant difference in cell loss was seen between groups at either time point alone. In the multivariate model, no significant increase in cell loss was associated with either tri-folding (3.7% less ECL; P = 0.051) or shipping (4.3% less ECL; P = 0.049). CONCLUSIONS: All techniques used resulted in clinically acceptable levels of ECL. Tri-folded tissue in a plastic cartridge did not result in ECL inferior to a scroll when prepared either immediately or preloaded for 48 hours.

Viability of Descemet Membrane Endothelial Keratoplasty Grafts Folded in the Eye Bank.

PURPOSE: Preloaded, trifolded grafts in Descemet membrane endothelial keratoplasty require transfer of the trifolding process from the corneal transplant surgeon to the eye bank technician. We sought to assess whether trifolding may be safely conducted by an eye bank technician with cell loss comparable to standard peeling and lifting. METHODS: A total of 10 grafts were stained, peeled, and transferred directly onto a bed of Calcein-AM and Amvisc Plus by an eye bank technician. Five grafts were removed and stained as a scroll, and 5 grafts were trifolded with the endothelium in before transfer. Photographs were acquired with an inverted fluorescence microscope, and image segmentation was performed. A t test was conducted to compare differences in endothelial cell loss across groups. RESULTS: Mean cell loss in the scroll group was 18.5% [95% confidence interval (CI): 15.2%-21.9%] compared with 7.6% of the trifolded group (95% CI: 1.7%-13.5%). A 2-tailed t test indicated decreased cell loss in the trifolded group (P = 0.013). CONCLUSIONS: Despite additional manipulation of the graft, trifolding of Descemet membrane and endothelium may be performed by an eye bank technician without significantly increased cell loss relative to graft preparation as a scroll.