Endothelial Keratoplasty Graft Research Articles

Peripheral Endothelial Cell Density in Descemet Membrane Endothelial Keratoplasty Grafts.

To compare the variation in corneal endothelial cell density (ECD) from the center to the periphery in unpeeled and peeled donor corneas and to determine the impact of eccentric trephining on total endothelial cells in Descemet membrane endothelial keratoplasty (DMEK) grafts. Mated donor cornea pairs were obtained. One cornea from each pair was peeled for DMEK, whereas the other was left unpeeled. Alizarin Red was used to stain the endothelial cells. High-resolution images at fixed magnification were obtained for the center, midperiphery (2.5 mm from the center), and the periphery (5 mm from the center). The cells were then counted, and ECD was calculated by a masked evaluator using ImageJ software. Regression analysis was then performed to evaluate the change in ECD as a function of radius (distance from the corneal center). The impact of eccentric trephining on total endothelial cells in a given DMEK graft was then calculated using numerical integration. Ten pairs of corneas were evaluated. ECD increased by 1.4% (40.0 cells/mm) (P = 0.03) for peeled corneas and 1.8% (51.5 cells/mm) (P < 0.01) for unpeeled corneas for each millimeter from the center. There was no difference between peeled and unpeeled corneas in the mean central (P = 0.98) or peripheral (P = 0.35) ECD. Based on the increase in ECD as a function of radius, eccentric trephining of a 7.5-mm DMEK graft by 2.25 mm yields 0.95% more total endothelial cells per graft. Corneal ECD increases from the center to the periphery in both peeled and unpeeled corneas. Eccentric trephining increases the number of transplanted endothelial cells per graft.

Supplementation of organ culture medium with dextran is not required in pre-stripped human donor tissue for DMEK surgery.

To assess corneal endothelial cell (CEC) quantity and quality in eye-bank prepared lamellar grafts for Descemet Membrane Endothelial Keratoplasty (DMEK) from organ cultured donor corneas that have not undergone de-swelling by media supplementation with dextran. Prior to graft preparation, corneas that had not undergone de-swelling (n = 30) were placed into fresh storage medium without dextran (KMI). Corneas in the control group (n = 30) were placed in dehydration medium containing 5% dextran (KMII). Subtotal stripping of Descemet's membrane (DM) was performed manually. Following graft preparation, 10 corneas of each group were cultured further in their respective medium for 24h, 72h, or 120h, respectively. Before and after DM stripping, as well as at the end of culture, CEC numbers were obtained. At the end of culture, CEC morphology was graded using a scoring system and CEC metabolism was assessed by detection of adenosine triphosphate. At 24h after DM stripping, mean CEC counts (in cells/mm2) were 2204 in corneas stored in KMII, and 2391 in corneas stored in KMI (p = 0.003). This corresponds to a mean relative CEC loss of 12.4% with dextran versus 9.7% without dextran (p = 0.04). At 72h, CEC counts were 1946 in KMII, and 2289 in KMI (p = 0.004). This corresponds to CEC loss of 23% with dextran versus 14% without dextran (p = 0.009). At 120h, CEC counts were 2047 in KMII, and 2230 in KMI (p = 0.14). This corresponds to CEC loss of 22.7% with dextran versus 17.2% without dextran (p = 0.14). Also, at 120h after DM stripping, 6/10 corneas fell below a threshold of 2000cells/mm2 if stored in medium containing dextran, versus 1/9 corneas if stored without dextran (p = 0.003). Morphological assessment of CEC quality revealed equal scores for cell polymorphism (median = 1), granulation (median = 0) and segmentation (median = 1) in all groups. Lower ATP/protein ratios were observed in corneas stored in medium without dextran at 24h (p < 0.001), 72h (p < 0.001), and 120h (p = 0.02). Abandoning the use of dextran in corneas destined for DMEK surgery leads to increased CEC counts and thereby serves to reduce tissue loss.

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

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. 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. 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. Endothelium-in DMEK grafts may be loaded into a plastic cartridge using a skill set similar to aspiration of a scroll.

Long-term observation of graft thickness and shape in Descemet stripping and automated endothelial keratoplasty.

To evaluate the thickness and shape of the posterior lamellar graft in Descemet stripping and automated endothelial keratoplasty after long-term observation. All patients who underwent Descemet stripping and automated endothelial keratoplasty including simple and triple Descemet stripping and automated endothelial keratoplasty between August 2009 and May 2014 were enrolled in this retrospective study. To assess postoperative thickness and shape of the Descemet stripping and automated endothelial keratoplasty graft, images of the graft taken at the center (C), mid-periphery at 4 mm optical zone (P1), and periphery at 6 mm optical zone (P2) at 1, 2, and 3 years postoperatively were obtained using anterior segment optical coherence tomography. C:P1 was 0.96, 0.96, and 0.95 at 1, 2, and 3 years postoperatively, respectively. C:P2 was 0.85, 0.84, and 0.83 at 1, 2, and 3 years postoperatively, respectively. There was a greater thinning of the central graft thickness compared with the peripheral graft thickness. The shapes of the posterior lamellar graft were variable, such as concave, asymmetrical, planar, irregular, and convex meniscus shapes. The most common shape was asymmetrical shape at 1 year postoperatively and concave at 2 and 3 years postoperatively. The most common shape of the posterior lamellar grafts was asymmetrical shape (38.18%) at 1 year postoperatively, followed by concave (34.54%), planar (20.00%), irregular (5.45%), and convex (1.81%) shapes. The most common shape was concave shape (44.44% and 57.14% for 2 and 3 years postoperatively, respectively), followed by asymmetrical (27.77% and 17.85%, respectively), planar (16.66% and 17.85%, respectively), and irregular (11.11% and 7.14%, respectively) shapes. In our study, concave meniscus was not the only observed shape of the graft. The shape and thickness of the graft did not stabilize even 3 years postoperatively in some patients. These findings should be taken into consideration in a combination of Descemet stripping and automated endothelial keratoplasty and cataract surgery.

Evaluation of the Suitability of Biocompatible Carriers as Artificial Transplants Using Cultured Porcine Corneal Endothelial Cells

ABSTRACTPurpose/Aim: Evaluating the suitability of bioengineered collagen sheets and human anterior lens capsules (HALCs) as carriers for cultivated porcine corneal endothelial cells (pCECs) and in vitro assessment of the cell-carrier sheets as tissue-engineered grafts for Descemet membrane endothelial keratoplasty (DMEK).Materials and Methods: pCECs were isolated, cultured up to P2 and seeded onto LinkCell™ bioengineered matrices of 20 µm (LK20) or 100 µm (LK100) thickness, and on HALC. During expansion, pCEC viability and morphology were assessed by light microscopy. ZO-1 and Na+/K+-ATPase expression was investigated by immunohistochemistry. Biomechanical properties of pCEC-carrier constructs were evaluated by simulating DMEK surgery in vitro using an artificial anterior chamber (AC) and a human donor cornea without Descemet membrane (DM).Results: During in vitro expansion, cultured pCECs retained their proliferative capacity, as shown by the positive staining for proliferative marker Ki67, and a high cell viability rate (96 ± 5%). pCECs seeded on all carriers formed a monolayer of hexagonal, tightly packed cells that expressed ZO-1 and Na+/K+-ATPase. During in vitro surgery, pCEC-LK20 and pCEC-LK100 constructs were handled like Descemet stripping endothelial keratoplasty (DSEK) grafts, i.e. folded like a “taco” for insertion because of challenges related to rolling and sticking of the grafts in the injector. pCEC-HALC constructs behaved similar to the DMEK reference model during implantation and unfolding in the artificial AC, showing good adhesion to the bare stroma.Conclusions: In vitro DMEK surgery showed HALC as the most suitable carrier for cultivated pCECs with good intraoperative graft handling. LK20 carrier showed good biocompatibility, but required a DSEK-adapted surgical protocol. Both carriers might be notional candidates for potential future clinical applications.

Learning curve of two common Descemet membrane endothelial keratoplasty graft preparation techniques

To compare the learning curve of two Descemet membrane endothelial keratoplast (DMEK) graft preparation techniques. Experimental study. Twenty paired donor corneoscleral rims. The corneas were randomized to DMEK peeling using the peripheral blunt dissection technique (n = 10) or the modified submerged cornea using backgrounds away (mSCUBA) technique (n = 10). Outcome measures included graft peeling time, surgeon's peeling difficulty grading (on a scale of 1 to 10, 1 being the easiest and 10 the hardest), number of tears, and percentage tissue loss. Average graft peeling time using the peripheral blunt dissection technique and the mSCUBA technique was 15.75 ± 4.01 minutes and 8.43 ± 3.26 minutes, respectively (p < 0.0005). The first 3 grafts' average peeling time was longer than the last 7 grafts: 19.14 ± 2.40 versus 14.21 ± 3.50 minutes in the peripheral blunt dissection technique (p = 0.06) and 12.36 ± 3.76 versus 6.67 ± 0.49 minutes in the mSCUBA technique (p = 0.016). In the latter, there were significantly fewer radial tears compared to the former: 1.5 ± 1.0 and 3.1 ± 1.9, respectively (p = 0.049). No tissue loss was noted in the mSCUBA group compared to one (10%) in the peripheral blunt dissection group. The average difficulty grading for the mSCUBA was significantly lower than the peripheral blunt dissection technique: 3.3 ± 1.9 and 5.8 ± 1.6, respectively (p = 0.024). Our study suggests a shorter learning curve with the mSCUBA technique for DMEK graft preparation, with shorter peeling time and fewer complications in comparison to the peripheral blunt dissection technique.

"Ghost DMEK" Technique: Circular Peripheral Staining of Descemet's Membrane Endothelial Keratoplasty Grafts.

To describe the ghost Descemet's membrane endothelial keratoplasty (DMEK) technique that allows for real-time evaluation of DMEK graft orientation, location, and folding status within the anterior chamber, by simple observation at the surgical microscope. This is a retrospective case series done in a university hospital. The study included 10 patients who underwent ghost DMEK surgery because of corneal endothelial insufficiency for Fuchs' endothelial dystrophy (N = 5), pseudophakic bullous keratopathy (N = 2), and failed previous posterior lamellar keratoplasty (N = 3). Cases were reviewed for feasibility of graft preparation, intra- and postoperative complications, postoperative clearing of the cornea, corrected distance visual acuity, and endothelial cell loss. All graft preparations were uneventful. The circular staining was readily apparent and did not fade away during surgery. Graft orientation and folding status were determined using only direct observation through the operating microscope. Complications included one intraoperative hyphema, one graft detachment requiring rebubbling, and one pupillary block on the first postoperative day, which was resolved surgically. At last visit, 8 of 10 eyes had visual acuity of 6/12 or better and 7 of 10 eyes had visual acuity of 6/8.5 or better. Mean cell loss rates 1 to 3 months postoperatively were 48% ± 17% (range, 22%-71%). All corneas were clear by the first to second postoperative month. Mean follow-up time was 3 months (range, 1-8 months). Peripheral staining of DMEK grafts (ghost DMEK) is a simple and effective method for graft visualization in the anterior chamber, obviating the need for trypan blue staining and other techniques and devices currently used for the identification of graft orientation and folding status, thus simplifying and shortening surgery.

Minimizing Endothelial Cell Loss Caused by Orientation Stamps on Preloaded Descemet Membrane Endothelial Keratoplasty Grafts.

To quantify endothelial cell loss (ECL) caused by orientation stamps on prestripped and preloaded Descemet membrane endothelial keratoplasty (DMEK) grafts, and to examine a method for reducing ECL using a smaller stamp. Ten prestripped and 10 preloaded DMEK grafts were prepared with S-stamps. Ten additional preloaded DMEK grafts were prepared with both an S-stamp and a smaller F-stamp in different paracentral areas of the graft. The footprint of each stamp was measured using ink on cardstock. DMEK grafts were stored in viewing chambers filled with 20 mL of Optisol-GS for 3 days at 4°C. ECL was quantified using Calcein-AM staining and FIJI Weka Segmentation. S-stamps on prestripped DMEK grafts contributed an average ECL of 1.1% ± 0.5% (range: 0.6%-2.2%) toward total graft damage, whereas S-stamps on preloaded DMEK grafts contributed approximately twice that amount (average ECL: 2.0% ± 0.7%, range: 1.3%-3.1%, P = 0.004). Overall ECL for prestripped grafts (average: 7.1% ± 3.3%, range: 3.3%-13.7%) and preloaded grafts (average: 11.3% ± 4.2%, range: 6.9%-19.4%) was similar to previous reports. The footprint of the S-stamp was approximately 45% larger than that of the F-stamp. In 10 preloaded grafts marked with both stamps, the S-stamp caused an average ECL of 1.9% ± 0.6% (range: 1.2%-3.2%), whereas the smaller F-stamp caused an average ECL of 1.0% ± 0.2% (range: 0.8%-1.4%, P = 0.0002). Loss of endothelial cells associated with graft-stamping was greater in preloaded tissue than in prestripped tissue and was less with a smaller F-stamp than with a larger S-stamp. Using a smaller stamp could help minimize ECL in prestripped and preloaded DMEK grafts.

Cross-Country Transportation Efficacy and Clinical Outcomes of Preloaded Large-Diameter Ultra-Thin Descemet Stripping Automated Endothelial Keratoplasty Grafts.

To evaluate the clinical outcomes of preloaded large-diameter ultra-thin grafts for Descemet stripping automated endothelial keratoplasty (UT-DSAEK) after cross-country shipment. A laboratory study in an eye bank and a clinical cohort study in an academic tertiary care center were performed. UT-DSAEK (9.5 mm diameter) grafts (n = 7) were prepared, loaded into a commercial device (iGlide; Eurobio, Les Ulis, France), preserved for 4 days at room temperature in transport medium, and analyzed. In a retrospective study, preloaded tissues (n = 39) for clinical use were prepared, transported from Italy to the United Kingdom, and surgically delivered into the eyes of patients undergoing UT-DSAEK. Central and peripheral endothelial cell density (ECD) and viability were measured before and after loading and storage of the grafts in the laboratory study. Clinically, best-corrected visual acuity, ECD before and at final follow-up, dislocation rate, primary graft failure, and surgical time were recorded. In the laboratory study, postcut central graft thickness was 93.3 ± 17.2 μm. ECD and cell mortality did not change significantly before and after preservation (P = 0.8). Cell loss after 4 days of preservation was 1.7% ± 1.6%. Clinically, 39 eyes of 39 patients at final follow-up showed a mean central graft thickness of 88 ± 22 μm and a best-corrected visual acuity of 0.34 ± 0.24 logMAR. Nine of 39 cases (23%) needed rebubbling, and 28% cell loss was observed at final follow-up. Large-diameter UT-DSAEK grafts can be prepared and preloaded in the eye bank using the iGlide and transported to the surgical center facilitating surgery for patients undergoing UT-DSAEK, potentially reducing tissue wastage, surgical time, and costs related to surgery.

The role of novel DMEK graft shapes in facilitating intraoperative unscrolling.

To evaluate whether the preparation of Descemet membrane endothelial keratoplasty (DMEK) grafts into various shapes affect their propensity to scroll as compared with the conventional circular graft design. Prospective randomized laboratory-based study, using nine pre-stripped DMEK corneal grafts unfit for transplantation. Each graft was trephinated into a standard circular 8-mm shape, stained with trypan blue, and then immersed in balanced salt solution (BSS). The width of the DMEK graft scroll was measured, and photographs were obtained. The graft was then cut into one of three pre-selected shapes (four peripheral punches, four radial cuts, Maltese cross). These newly shaped grafts were then replaced in BSS and again photographed. The scroll widths, as well as pre- and post-preparation scroll width-to-height ratios, were calculated and compared. Nine pre-stripped DMEK corneal grafts (mean donor age ± SD 73.1 ± 9.3years, range 58-85years) were included. The mean pre-cut scroll widths for the three selected shapes were statistically similar. Following graft preparation into their assigned shapes, the mean post-preparation scroll widths (and corresponding change from pre-preparation measurements) were 1.73 ± 0.16mm (- 3.6%) for the 4-peripheral punches, 2.59 ± 0.35mm (+ 0.2%) for the 4-radial cuts and 4.13 ± 0.63mm (+ 20.1%) for the Maltese cross (P = 0.0013). Therefore, the Maltese cross design resulted in a wider scroll than its pre-preparation control. Certain DMEK graft shapes may be less prone to scrolling than others. The Maltese cross graft design scrolled less tightly than the other experimental graft shapes. Future studies may elucidate intraoperative scrolling behavior of these DMEK graft shapes within the anterior chamber.

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

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. 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. 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). 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.

Tectonic epikeratoplasty with ethanol-stored donor corneas.

To evaluate the efficacy and outcomes of tectonic epikeratoplasty with use of ethanol-preserved corneal grafts for the management of perforated corneal melts. The present retrospective case series includes 10 eyes which underwent tectonic epikeratoplasty for perforated corneal melts. The stromal remainders of Descemet membrane endothelial keratoplasty (DMEK) and Descemet stripping automated endothelial keratoplasty (DSAEK) graft preparation were stored in 95% ethanol and used as emergency tectonic grafts for restoring globe integrity after sterile and infectious perforated corneal melts. In 6 cases with subtotal corneal melt, DMEK remainders (endothelium-denuded corneoscleral buttons) were used for 'limbus to limbus' tectonic epikeratoplasty and in 4 cases DSAEK remainders (anterior stroma) were used to seal focal perforated melts. Graft storage time was 5.1 ± 4.9 (ranging from 0.5 to 17) months. The surgeries were successful in all cases with restitution of the globe integrity. During the postoperative course 4 cases developed a graft melt (corneoscleral button for limbus to limbus tectonic epikeratoplasty, n = 3; lamellar patch, n = 1) within 2-6months after the initial procedure. Three patients underwent successful repeat tectonic epikeratoplasty. In the fourth case of graft melt the globe was enucleated due to underlying expulsive haemorrhage and severe pain. The short-term results of the present case series suggest that the use of ethanol-stored stromal remainder of donor corneas after endothelial keratoplasty is an efficient temporary measure for tectonic restoration of perforated corneas.

Clinical Outcomes of Preloaded Descemet Membrane Endothelial Keratoplasty Grafts With Endothelium Tri-Folded Inwards

To evaluate the initial outcomes and complications of Descemet membrane endothelial keratoplasty (DMEK) using donor tissues tri-folded with the endothelium inwards, preloaded at the Eye Bank, and delivered with bimanual pull-through technique. Prospective, noncomparative, interventional case series. Setting: Eye bank and tertiary care eye department. Forty-six consecutive eyes of 41 patients with Fuchs endothelial dystrophy with or without cataract operated between November 2016 and March 2017. DMEK tissues prepared with SCUBA technique and punched to a diameter of 8.25mm were preloaded with the endothelium tri-folded inwards in an intraocular lens (IOL) cartridge with a 2.2-mm opening filled with the same tissue culture medium contained in the vial used for shipment to the surgeon. Standardized DMEK was performed as a single procedure (n= 15) or in combination with phacoemulsification and IOL implantation (n= 31) within 48 hours from preparation using a bimanual pull-through technique. Preparation and surgical times, intraoperative and postoperative complications, best spectacle-corrected visual acuity (BSCVA), endothelial cell density (ECD), and graft detachment rate. Preparation time averaged 26.2 ± 4.1minutes (range 17-36minutes), while the surgical time from opening of the stoppers to air fill of the anterior chamber never exceeded 9minutes (range 3-9minutes). Surgery was uneventful in all cases. Postoperative complications included graft detachment in 9 of 46 cases (19.6%), successfully managed in all cases by single rebubbling within 6days from surgery, and glaucoma irresponsive to conservative treatment in 1 of 46 cases (2.1%). In all eyes without comorbidities (35 of 40 eyes) BSCVA was 20/25 (0.097 logMAR) or better as early as 3months after surgery. Six months postoperatively, ECD was available in 24 of 25 eyes with an endothelial cell loss calculated as a percentage of the preoperative value determined at the eye bank (ranging from 2500 to 2800 cells/mm2) of 29.5% ± 14.8% (range 8.3%-52.1%). Delivering a preloaded DMEK tissue, tri-folded with the endothelium inwards, minimizes surgical time and costs without negatively affecting the outcomes ofthe procedure.

Biomechanical evaluation of central and peripheral Descemet’s membrane endothelial graft

Corneal endothelial transplant is the gold-standard treatment in cases of corneal endothelial cellular dysfunction. Preparation, delivery, and unfolding of the graft are technically demanding. We assessed the biomechanical behavior of Descemet’s membrane to better understand Descemet’s membrane endothelial keratoplasty(DMEK) graft behavior, and to select the right diameter and donor age graft . The biomechanical behavior was tested using atomic force microscopy (AFM) on five corneas unsuitable for transplantation. The peripheral cornea was found to be stiffer than the central cornea (3171.89 MPa and 2837.20 MPa, respectively). The elastic modulus of both the central and peripheral cornea exhibited a trend to decrease with age. In addition, the central cornea becomes stiff er than the peripheral cornea in older patients, while the peripheral cornea was stiff er in younger patients. AFM is a suitable technique for evaluating biomechanical behavior of DMEK graft s. One interpretation of this varied behavior is that the type and quantity of collagen changes with age and location.

The feasibility and efficacy of preparing porcine Descemet's membrane endothelial keratoplasty (DMEK) grafts by two techniques: An ex-vivo investigation for future xeno-DMEK.

Descemet's membrane endothelial keratoplasty (DMEK) might be a promising technique for future xeno-corneal transplantation due to its ultrathin graft, extremely low rejection occurrence, suture-free graft fixation, and minimal immunosuppressive regime usage. The aim of this study is to explore the feasibility and efficacy of preparing porcine DMEK grafts by 2 techniques and investigate the graft ultrastructure. Two mainstream techniques, mechanical stripping technique and liquid bubble technique, were modified to prepare the porcine DMEK grafts. In all, 40 corneas harvested from WZS-pigs (aged 10-12months) were subjected to the techniques (20 corneas for each technique). The success rate, time consumption, and endothelial cell density (ECD) before and after preparation were recorded and compared between the 2 techniques. And the ultrastructure of the porcine DEMK graft was investigated by transmission electron microscope. In addition, 9 WZS-pigs with different ages were sacrificed to explore the correlation between the thickness of Descemet's membrane and porcine age. After modifying several technical details, the porcine DMEK grafts were successfully prepared by either mechanical stripping technique or liquid bubble technique, and the mark technique to distinguish the 2 sides of the graft was also explored. In all, 13 DMEK grafts (65%) were prepared successfully by the mechanical stripping technique, whereas 14 successful cases (70%) were prepared by the liquid bubble technique. The success rates between the 2 techniques showed no significant difference (P=.847). However, the mechanical stripping technique was significantly time-consuming when compared with the liquid bubble technique (P<.0001). The ECDs reduced significantly after preparation no matter what techniques were used (P<.0001), but the ECD after the liquid bubble preparation was significantly higher than the ECD after mechanical stripping (P=.032). The ECD reduction positively correlated to the time consumption for both mechanical stripping technique (P=.0014, R2 =0.621) and liquid bubble technique (P=.013, R2 =0.412). The ultrastructure showed the graft was comprised of stromal residuals, non-banded layer, and endothelial layer. Unlike human Descemet's membrane (DM), anterior banded layer was not observed. The thickness of porcine DM increased with the age, and a significant positive correlation between them was found (P<.0001, R2 =0.949), and the predict equation was Y=0.3764*X+7.378 (Y indicates the thickness, whereas X indicates the age). Porcine DMEK grafts could be prepared either by mechanical stripping technique or liquid bubble technique, and the liquid bubble technique seems superior over the mechanical stripping technique regarding time consumption and ECD preservation. Although there are several technical barriers to overcome, xeno-DMEK might be a promising direction for future xeno-corneal transplantation.

Descemet Membrane Endothelial Keratoplasty with Irregular-Edged Graft: A Salvage Method for Large Radial Graft Tears.

Large radial tears of donor Descemet membrane (DM) during the preparation of Descemet membrane endothelial keratoplasty (DMEK) grafts can make the trephination stage impossible because of small graft diameter. This results in irregular-edged grafts. In this study, we report two pseudophakic bullous keratopathy patients who underwent DMEK surgery with irregular-edged Descemet membrane (DM) grafts. Main outcome measures were preoperative and postoperative 1-, 3-, and 6-month best corrected visual acuity (BCVA), endothelial cell density (ECD) and central corneal thickness (CCT). Intraoperative and early postoperative complications were also evaluated. Both irregular-edged grafts were successfully implanted into the anterior chamber, unfolded, and attached to the posterior corneal stroma. Patients’ BCVA at 6 months was 1.0 (Snellen equivalent: 20/20) and 0.6 (Snellen equivalent: 20/32) respectively. Decrease in ECD at the last visit was 27% and 25%. CCT decreased from 723 μm and 850 μm to 530 μm and 523 μm, respectively. No intraoperative complications occurred except for the large radial Descemet membrane graft tears that developed during donor DM stripping. None of the cases needed a rebubbling procedure postoperatively. We have demonstrated that irregular-edged DM grafts can be successfully implanted for DMEK surgery with good clinical outcomes.

New method for the preparation of Descemet membrane endothelial keratoplasty graft with Hessberg-Barron negative pressure trephine

Endothelial keratoplasty is the most popular surgical procedure in recent years. In this article the method for preparation of Descemet membrane endothelial keratoplasty graft is modified. The new method is compared with the traditional method. Key words: Descemet membrane endothelial keratoplasty(DMEK); Preparation, graft, donor; Mo-dification

Measuring Endothelial Cell Loss on DMEK Grafts After Transplantation in Human Cadaveric Whole Eyes: Description of the Technique and Pilot Study.

To describe a novel method for analyzing Descemet membrane endothelial keratoplasty (DMEK) graft damage after implantation into human cadaveric donor eyes and to compare results achieved by performing DMEK with a surgeon's long-established technique compared with those of an unfamiliar technique. Eight DMEK grafts were implanted into previously frozen human cadaveric eyes. Four grafts were implanted using a Straiko injector and tap technique familiar to the surgeon (C.S.S., 3-yr experience), and 4 grafts were implanted using the Tan EndoGlide and "donor mat device" pull-through technique new to the surgeon. After implanting a DMEK graft and attaching it to the recipient stroma with an air bubble tamponade, the corneoscleral cap was "recovered" from the cadaveric globe using standard techniques. The DMEK graft was stained with Calcein-AM. After staining, a 9.5-mm stromal "carrier button" was punched, and the carrier and graft were transferred to a microscope slide. Grafts were imaged and analyzed using FIJI trainable segmentation. Donor graft characteristics were similar between both groups. Grafts implanted using the surgeon's routine technique showed an average endothelial cell loss (ECL) of 31% ± 4% (n = 3). Grafts implanted using the technique unfamiliar to the surgeon showed an average ECL of 47% ± 24%, but with a trend toward improvement (1 = 76%, 2 = 65%, 3 = 32%, 4 = 17% ECL). Our proof-of-principle experiment shows that this imaging approach enables quantification of ECL caused by different instruments and surgical techniques after graft implantation. We have used this method to visualize the learning curve of 1 surgeon when learning a new surgical technique.

Cumulative Endothelial Cell Loss in Descemet Membrane Endothelial Keratoplasty Grafts From Preparation Through Insertion With Glass Injectors.

Evaluation of cumulative Descemet membrane endothelial keratoplasty endothelial cell loss (ECL) from preparation through injection using 2 different glass injectors. Eighteen Descemet membrane endothelial keratoplasty grafts with "S" stamps were prepared by eye bank technicians. Nine grafts were assigned to injection with a modified glass Jones tube injector with a 2.4-mm opening and 9 were assigned to injection with the DORC glass pipette injector (<1.5-mm opening). The grafts were prepared and loaded into the injectors using the standard surgical technique, ejected onto a bed of viscoelastic on a glass slide, and unscrolled using viscoelastic. The grafts were stained with the vital dye Calcein-AM, then digitally imaged and analyzed using FIJI. The percentage of ECL was calculated by measuring the area of nonfluorescent pixels and dividing it by the total graft area pixels. A statistical comparison was performed using a 2-tailed unpaired t test. Grafts injected using the DORC injector versus Jones tube injector had ECL of 29.2% ± 8.5% [95% confidence interval (CI)] versus 23.0% ± 5.1% (95% CI), respectively. This difference was not statistically significant (P = 0.17); however, the patterns of ECL on the grafts was different between injectors. Peripheral ECL caused by trephination and surgeon grasp sites accounted for 7.5% ± 1.2% (95% CI). There was no statistical difference in ECL between the 2 injectors. There were characteristic differences in patterns of ECL seen between injectors, which may be clinically relevant and indicate the types of stress that grafts are exposed to during passage through various injectors.

Outcomes of Descemet Membrane Endothelial Keratoplasty in Eyes With a Previous Descemet Stripping Automated Endothelial Keratoplasty Graft.

To analyze the outcomes of Descemet membrane endothelial keratoplasty (DMEK) in eyes with previous Descemet stripping automated endothelial keratoplasty (DSAEK). This retrospective interventional case series included 26 eyes (26 patients) that underwent DMEK to replace a previous DSAEK graft with at least 6 months of follow-up. The outcome measures were indications for surgery, best spectacle-corrected visual acuity (BSCVA), endothelial cell density, rebubbling rate, rejection, and failure. Patient age was 71.9 ± 12.6 years. The average follow-up time after DMEK was 15.1 ± 10.6 months. Indications for DMEK were DSAEK graft failure (69%) and a DSAEK suboptimal visual outcome (31%). BSCVA of the entire cohort (n = 26) improved from 1.33 ± 0.78 logMAR preoperatively to 1.04 ± 0.78 and 1.06 ± 0.89 logMAR at 6 months postoperatively and at the last follow-up, respectively (P = 0.019 and P = 0.033). BSCVA among eyes without visual comorbidities (n = 13) improved from 0.84 ± 0.50 logMAR preoperatively to 0.55 ± 0.47 and 0.51 ± 0.49 logMAR at 6 months postoperatively and at final follow-up, respectively (P = 0.023 for both). Of these eyes, 84.6% had improvement in BSCVA at 6 months postoperatively and at last follow-up. In the subgroup of 8 eyes with DMEK for suboptimal visual outcomes after DSAEK, BSCVA improved from 0.81 ± 0.44 to 0.52 ± 0.35 logMAR at final follow-up (P = 0.024). When excluding eyes with visual comorbidities, BSCVA of this subgroup (n = 5) improved from 0.54 ± 0.32 to 0.36 ± 0.25 logMAR at final follow-up (P = 0.038). BSCVA of this subgroup at 6 months postoperatively was not significantly different from preoperative BSCVA, when including visual comorbidities (n = 8, 0.75 ± 0.60 logMAR, P = 0.79) and when excluding visual comorbidities (n = 5, 0.40 ± 0.28 logMAR, P = 0.621). Endothelial cell density decreased from 2753 ± 307 cells/mm to 1659 ± 655 cells/mm 6 months after surgery (39.7% loss, P = 0.005). Three eyes (11.5%) required rebubbling, and 5 eyes (19.2%) had secondary graft failure at 2 to 20 months. DMEK is effective for replacing previous DSAEK not only for graft failure but also for suboptimal visual outcomes.