Deep Anterior Lamellar Keratoplasty Using Dehydrated versus Standard Organ Culture-Stored Donor Corneas: Prospective Randomized Trial.

PURPOSE: To compare the outcomes of deep anterior lamellar keratoplasty (DALK) using dehydrated versus standard organ culture-stored donor corneas for eyes with keratoconus. DESIGN: Prospective, randomized, single-center trial conducted in Italy. PARTICIPANTS: Adult patients (age ≥ 18 years) with keratoconus scheduled for elective DALK. METHODS: Patients undergoing successful type 1 bubble pneumatic dissection using a standard DALK technique were randomized during surgery to receive either dehydrated (n = 30) or standard organ culture-stored (n = 30) donor corneas. MAIN OUTCOME MEASURES: The primary study outcome was best spectacle-corrected visual acuity (BSCVA) 12 months after surgery. Secondary outcomes were refractive astigmatism (RA), endothelial cell density (ECD), and complication rates. RESULTS: Postoperative BSCVA did not significantly differ between groups at both time points: mean difference at 6 months was 0.030 logarithm of the minimum angle of resolution (logMAR; 95% confidence interval [CI], -0.53 to 0.10 logMAR; P = 0.471) and at 12 months was -0.013 logMAR (95% CI, -0.10 to 0.08 logMAR; P = 0.764). No significant differences between groups were observed in terms of postoperative RA and ECD at all time points. In the first 3 days after DALK, an epithelial defect was present in 10 patients (33%) in the organ culture cornea group and in 29 patients (97%) in the dehydrated cornea group. Complete re-epithelialization was achieved by day 7 in all patients (100%) in both groups. CONCLUSIONS: The study provides evidence that the use of dehydrated corneas is noninferior to the use of standard organ culture donor corneas for DALK. Corneal tissue dehydration represents a viable solution that can allow long-term cornea preservation and avoid wastage of unused corneas. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Hemi-Ultrathin Descemet Stripping Automated Endothelial Keratoplasty (Hemi-UT-DSAEK) Using Pediatric Donor Corneas: A Case Series.

OBJECTIVE: We sought to evaluate the clinical outcomes of hemi-UT-DSAEK grafts from the pediatric donor corneas of patients affected by Fuchs Endothelial Corneal Dystrophy (FECD). METHODS: A prospective, interventional case series was conducted at the Ophthalmology Department of Venice Civil Hospital and the Veneto Eye Bank Foundation (Venice, Italy). Six eyes of six patients affected by FECD received large-diameter, semicircular hemi-UT-DSAEK grafts obtained from three pediatric donor corneas using the standard pull-through method. Endothelial cell density (ECD), central corneal thickness (CCT), best-corrected visual acuity (BCVA) and intraoperative and postoperative complications were recorded at different time intervals up to 12 months. RESULTS: The average donor age was 64.6 ± 8.6 years, and the pre-operative ECD was 3266 ± 225 cells/mm2. At 12 months postoperatively, the average ECD was 1376 ± 509 cells/mm2 with a mean decrease of 56.8 ± 19.1% from the preoperative donor count. At 12 months, four out of six eyes had significantly improved and reached a BCVA of ≥20/25 (Snellen equivalent). The mean CCT significantly decreased from 788 ± 138 µm before surgery to 576 ± 30 µm at 12 months postoperatively (p < 0.01). CONCLUSIONS: Hemi-UT-DSAEK grafts using pediatric donor corneas are surgically feasible and can provide similar clinical outcomes compared to conventional UT-DSAEK. Transplanting pediatric donor tissues with high ECD into two patients could potentially increase the donor tissue pool to treat endothelial disease.

Effect of Liposomal-Lactoferrin-Based Eye Drops on the Conjunctival Microflora of Patients Undergoing Cataract Surgery.

INTRODUCTION: Postoperative endophthalmitis is typically caused by the patient's conjunctival bacterial flora. Povidone iodine solution (5%) is used perioperatively to obtain periocular and ocular antisepsis. However, an adjunctive prophylaxis procedure could further help control the conjunctival microbial load. Considering the increase in antibiotic resistance, a progressive shift toward alternative methods would be desirable. Somilux® eye drops (Alfa Intes, lactoferrin-based eye drops) are medical devices containing liposomal lactoferrin (LF). This study evaluates the effects on conjunctival microflora of LF-based eye drops used in the preoperative phase in patients scheduled for cataract surgery. METHODS: LF-based eye drops or a vehicle solution (water solution) were instilled 4 times a day starting 3 days before cataract surgery. Before the therapy (T0) and at the time of surgery (T1), a conjunctival swab was performed in both eyes and processed to detect microbial growth, microbiological isolation, and species identification. The outcome was the quantification and characterization of the local microbial flora before and after using LF-based or vehicle-based eye drops. Safety of the treatments was also evaluated. RESULTS: 88 eyes of 44 patients (mean [± SD] age 75 [± 12.6] years) were enrolled. At baseline, 54 conjunctival swabs showed only saprophytic flora, 27 showed only potential pathogenic flora, and seven showed both of them. LF-based eye drops reduced the proportion of potentially pathogenic bacteria (36% at T0 vs. 9% at T1, p = 0.008) compared with the vehicle (41% at T0 vs. 55% at T1, p = 0.302) without altering the physiological ocular microbial composition. No adverse events have been reported. CONCLUSION: Our findings provide a novel contribution to the scientific knowledge on the role of LF in the ophthalmic field, supporting the use of LF-based eye drops as a safe and selective treatment to improve the ocular surface physiological defenses and control the bacterial ocular surface contamination prior to cataract surgery.

Stromal peeling for deep anterior lamellar keratoplasty in a post-penetrating keratoplasty eye with hematocornea.

Purpose: To present a case of hematocornea occurring in a post-penetrating keratoplasty (PK) eye and to report the outcomes of deep anterior lamellar keratoplasty (DALK) performed by simple stromal peeling. Observations: A 45-year-old female presented with hematocornea in the left eye that previously underwent PK 26 months prior for keratoconus. Clinical examination revealed a dense reddish-brown opacity within the PK graft which was associated with deep corneal neovascularization. Over 6 months, intracorneal hemorrhage developed a rust-colored appearance with minimal clearing. DALK was performed using the stromal peeling technique for post-PK eyes. Briefly, a dense partially organized hemorrhage was identified at the natural plane of separation, as confirmed by ex vivo histologic examination; after peeling of the deep corneal stroma and evacuation of the intracorneal hemorrhage, the residual bed appeared akin to pre-Descemet's layer-Descemet membrane-endothelium complex. One year after DALK, the graft remained clear with ECD of 1034 cells/mm2. Conclusions and Importance: Intracorneal hemorrhage is a rare but potentially sight-threatening complication following PK. Using the stromal peeling technique, DALK can be attempted to preserve functional endothelium in post-PK eyes. In the presence of a dense intracorneal hemorrhage, the spread of erythrocytic debris within the stroma can guide deep lamellar cleavage.

Results of ultrathin Descemet stripping automated endothelial keratoplasty with donor corneas preserved in synthetic organ culture media.

PURPOSE: To evaluate outcomes of ultrathin Descemet stripping automated endothelial keratoplasty (UT-DSAEK) with donor corneas preserved at 31°C in Cornea Syn®, a medium formulated with recombinant human serum albumin (rHSA) to replace foetal calf serum, and deswelled-transported in the xeno-free medium Cornea Trans®. METHODS: Prospective, multicentre, open-label study. We evaluated the endothelial cell loss (ECL) as the percentage variation of the endothelial cell density (ECD, cells/mm2) between 6 and 12 months after surgery, corneal transparency and thickness at 12 months, and adverse events within 12 months. Endothelial lenticules of mean 89 µm, ECD ≥ 2300 cells/mm2, minimum signs of cell mortality or morphology alterations, were dissected by microkeratome in the eye bank, and grafted in patients ≥ 18 years without corneal neovascularisation, conjunctivalization, or blinking impairment. RESULTS: Thirty-five patients underwent UT-DSAEK, 3 showed primary failure, 1 late failure, and 2 skipped the 6-month visit. We analysed data from 29 patients, 27 with Fuchs endothelial corneal dystrophy (FECD) and 2 with pseudophakic bullous keratopathy (PBK). The median ECL between 6 and 12 months was 2.6% (p = .054, CI 0 to 12.5) and the absolute mean (SD) was 158.4 (364.1) cells/mm2. After 12 months, 96.5% of corneas were clear, with mean pachymetry of 585.9 (50.4) µm. CONCLUSIONS: The ECL rate after UT-DSAEK match overall that observed in DSAEK or UT-DSAEK models of endothelial survival and the overall safety compared that reported for similar follow-up. Corneas maintained in Cornea Syn® and Cornea Trans® did not affect the ECD and functional outcomes of UT-DSAEK up to 12 months.

Cryopreservation of human amniotic membrane for ocular surface reconstruction: a comparison between protocols.

PURPOSE: To compare the effects on adhesive and structural properties of newer preservation conditions to those obtained with an established, standardized protocol (dimethyl sulfoxide at -180 °C). In attempt to simplify and enhance the safety of the procedure, we tested dextran-based freezing medium and a dry condition (no medium) at temperatures of -80 °C. METHODS: Five patches of human amniotic membrane were obtained from three different donors. For each donor, five preservation condition were tested: dimethyl sulfoxide at -180 °C, dimethyl sulfoxide at -80 °C, dextran-based medium at -180 °C, dextran-based medium at -80 °C and dry freezing at -80 °C (no medium). At the end of four months storage period, adhesive properties and structure were analyzed. RESULTS: None of the newer preservation protocols showed differences in adhesive and structural properties of the tissues. The stromal layer always kept its adhesiveness, while both structure and basement membrane were not altered by any the preservation protocol. CONCLUSIONS: Switching from liquid nitrogen cryopreservation to -80 °C would reduce manipulation, simplify the procedure, making it also cheaper. The use of dextran-based freezing medium or no medium at all (dry condition) would avoid the potential toxicity of the dimethyl sulfoxide-based freezing media.

Gram stain and addition of amphotericin B to improve the microbial safety of human donor corneas.

To determine the effectiveness of two methods to improve the microbial safety of human corneas preserved in organ culture. We compared the number of positive preservation solutions of corneas in organ culture in which the initial short-term hypothermic corneal maintenance solution was supplemented with amphotericin B 2.5 µg/mL and the historical data of microbial test results (2015-2019). In addition, we appraised the efficacy of Gram stain to detect bacterial or fungal contamination in the organ culture solutions of corneas from at-risk donors compared to the culture tests of corneas from not-at-risk donors. Statistical analysis was performed using STATA and statistical significance set at p < 0.05. The number of positive culture tests after preservation was 15 (0.5%) in 2020 compared to a mean of 37 (1.2%) in the period 2015-2019 (p < 0.01), with 10 (1.0%) positive samples in the cohort of 998 corneas from at-risk donors and 5 (0.2%) in the 2046 corneas from not-at-risk donors (p < 0.01). All corneas from at-risk donors tested positive at Gram stain and the results were available 1-3 days before those of the conventional culture tests. Amphotericin B supplementation in the short-term maintenance solution markedly reduced the number of positive microbial tests after organ culture and the early detection of contaminants, including slow-growing microorganisms, by Gram stain before the standard culture results. This meant fewer corneas being discarded and a greater likelihood of preventing post-graft infections.

23†Cryopreservation of human amniotic membrane (HAM) for ocular surface reconstruction: a comparison beetween protocols.

PURPOSE: In the past decades, the human amniotic membrane has been largely applied for several surgical and non-surgical procedures. It has been farther demonstrated that both hAM and cornea share similar patterns of expression of structural components of the basement membrane (like laminin 5 and collagen IV) making hAM an useful tissue for ocular surface reconstruction. Since 1996 in fact, amniotic membrane transplantation has been applied to a large number of ocular surface diseases including Stevens-Johnson syndrome, pterygium, corneal ulceration, ocular surface reconstruction after chemical/thermal burns and in the reconstruction after excision of ocular surface neoplasia. During the previous decades, hAM has achieved a pivotal role in regenerative medicine too.The possibility to preserve human amniotic membrane, without affecting membrane's features, has become pivotal, allowing virological and microbiological analyses to be carried out before grafting. The purpose of the present study is to investigate an easier and cheaper protocol for human amniotic membrane preservation without affecting its properties and structure, ensuring the safety profile of the tissue. We compared the effects on adhesive and structural properties of newer preservation conditions to those obtained with an established, standardized protocol (dimethyl sulfoxide at -160°C). In attempt to simplify and enhance the safety of the procedure, we tested dextran-based freezing medium and a dry condition (no medium) at temperatures of -80°C. METHODS: Five patches of human amniotic membrane were obtained from three different donors. For each donor, five preservation condition were tested: dimethyl sulfoxide at -160°C, dimethyl sulfoxide at -80°C, dextran-based medium at -160°C, dextran-based medium at -80°C and dry freezing at -80°C (no medium). At the end of four months storage period, adhesive properties and structure were analyzed. RESULTS: None of the newer preservation protocols showed differences in adhesive and structural properties of the tissues. The stromal layer always kept its adhesiveness, while both structure and basement membrane were not altered by any the preservation protocol. CONCLUSIONS: Switching from liquid nitrogen cryopreservation to -80°C would reduce manipulation, simplify the procedure, making it also cheaper. The use of dextran-based freezing medium or no medium at all (dry condition) would avoid the potential toxicity of the dimethyl sulfoxide-based freezing media.

Efficacy of a New Commercial Ocular Spray Containing Oftasecur Citrus Extract for Reducing Microbial Load in the Conjunctiva of Patients Receiving Intravitreal Injections.

INTRODUCTION: The control of conjunctival microbial load is crucial for patients receiving intravitreal injections (IVTs) in order to reduce the risk of endophthalmitis. The purpose of this work was to assess the antimicrobial activity of a new commercial ocular spray containing Biosecur citrus extract (Oftasecur®, Off Health, Florence, Italy). METHODS: This prospective cross-sectional pilot study included patients receiving IVTs who were instructed to apply Oftasecur spray onto the eye to be injected four times daily starting 4 days before surgery. The contralateral eye was considered the control. A conjunctival swab for microbiological analysis was performed in both eyes before starting study treatment and at the time of the injection. The Brief Ocular Discomfort Inventory (BODI) questionnaire was administered to patients based on an 11-point scale (0 for no discomfort and 10 for maximum discomfort). RESULTS: Thirty patients (15 male, 15 female; mean age 64.7 ± 11.6 [standard deviation, SD] years) were included. Before starting treatment, 53.3% of the total eyes tested positive during the microbiological analysis. After the treatment period, only 20% of the eyes tested positive at the time of injection, showing a significant reduction in the microbial load (p < 0.01). Moreover, in the treated arm, the positive swabs before and after the prophylactic treatment with Oftasecur ocular spray showed a significant reduction (from 70.4% to 29.6%; p = 0.003, McNemar's test). Oftasecur ocular spray was well tolerated, with an average BODI score of 1.2 (± 0.70 SD). CONCLUSION: Oftasecur ocular spray showed antimicrobial activity that significantly reduced the microbial load in patients receiving intravitreal injections. Therefore, it may have a role in the prophylaxis of infection in the setting of IVTs.

Ultrastructural Analysis of Rehydrated Human Donor Corneas After Air-Drying and Dissection by Femtosecond Laser.

Purpose: To evaluate the efficiency of femtosecond laser (FSL) incision of rehydrated human donor corneas after air-drying and its effects on corneal structure. Methods: We compared the rehydrated and fresh-preserved corneas by microscopy following Victus-Tecnolas FSL treatment for straight-edge anterior lamellar keratoplasty (ALK). The corneas were dehydrated at room temperature under a laminar-flow hood. Results: To obtain the horizontal cut in rehydrated corneas, we increased the FSL pulse energy to 1.2 µJ from 0.80 µJ applied for the fresh corneas and obtained a clear-cut separation of the lamellar lenticule cap from the corneal bed. Light microscopy showed regular arrangement of stromal collagen lamellae, with spaces in between the fibers in the corneal stroma in the fresh and the rehydrated corneas, but the uppermost epithelial layers in the rehydrated corneas were lost. Transmission electron microscopy (TEM) revealed no signs of thermal or mechanical damage to the corneal structure. The epithelial basal membrane and Bowman's layer maintained their integrity. The epithelial basal layer and cells were separated by large spaces due to junction alteration in the rehydrated corneas. There were gaps between the lamellar layers in the stroma, especially in the rehydrated corneas. Keratocytes displayed normal structure in the fresh corneas but were devoid of microorganules in the rehydrated corneas. Minor irregularities were observed in the vertical incision and the horizontal stroma appeared smooth on scanning electron microscopy. Conclusion: The corneal stroma of rehydrated corneas maintained morphology and integrity, while corneal cellular components were generally altered. When corneas are intended for FSL-assisted ALK, effective stromal bed incision is best achieved at a laser power higher than that currently adopted for fresh corneas.

Next-generation sequencing for the detection of microorganisms present in human donor corneal preservation medium.

OBJECTIVE: To detect the presence of microorganisms in the storage media of human donor corneas using next-generation sequencing method. METHODS: Seven samples from organ culture (OC) group (Cornea Max, Eurobio, Les Ulis, France) with one control (sterile media without any cornea) and seven samples from hypothermic storage group (Cornea Cold, Eurobio) with one control were used for this study. The corneas were placed in the respective storage media for 14 days before collecting the samples. Storage media (2 mL) from each sample were collected in RNAase-free tubes and shipped for ribosomal RNA sequencing of 16 S and 18 S. Simultaneously, another 1 mL of media sample was used for conventional diagnostic method (CDM) using Bactec instruments. RESULTS: In both, OC and hypothermic storage and control samples, the most abundant genera were Pseudomonas, Comamonas, Stenotrophomonas, Alcanivorax, Brevundimonas and Nitrobacter. Acidovorax, Acetobacter and Hydrogenophilus were detected mostly in the hypothermic storage group. The most abundant fungal pathogen detected belonged to the genus Malassezia, which was found in both the storage conditions. CDM was negative for microorganisms in all the samples. CONCLUSION: Metagenomics provides full taxonomic profiling of the detected genomic material of the organisms and thus has the potential to deliver a much wider microbiological diagnostic approach than CDM. The costs and turn-around time need to be reduced, and; the detection of viable organisms would help this technology to be introduced into routine clinical practice.

Asymptomatic Infection in Decompensated Full-Thickness Corneal Grafts Referred for Repeat Penetrating Keratoplasty.

PURPOSE: We report a case series of asymptomatic infections affecting failed corneal grafts in patients referred for repeat penetrating keratoplasty (PK). METHODS: In this retrospective, noncomparative, interventional case series, we reviewed the medical records of all repeat PK procedures performed at Villa Serena-Villa Igea private Hospitals (Forlì, Italy) between January 2011 and March 2016. Specifically, preoperative and postoperative slit-lamp examinations, and the results of histological and bacteriological examinations, were noted. RESULTS: Fifty-three repeat PKs were performed in the study period. All patients were referred because of long-standing graft decompensation with stromal scars or surface irregularities, thus unsuitable for endothelial keratoplasty. None was referred because of presumed infection. Histological examination of the explanted buttons showed the presence of microorganisms of various types in 7 eyes. Cultures were positive in 4 of these cases and in one additional case Staphylococcus aureus was grown in culture, but was not seen in the histology specimen. None of the patients presented with unusual pain, tearing, or discomfort. Preoperative abnormal clinical findings included epithelial defect (n = 6), focal whitening of corneal stroma (n = 5), crystalline keratopathy (n = 1), and an elevated pigmented lesion (n = 1). After repeat PK, recurrence of the infection was seen in 5 of 7 (71%) cases, 2 of which required a third PK procedure. CONCLUSIONS: Apparently quiet eyes with failed PK can harbor slow-growing asymptomatic infection. An epithelial defect in a failed PK graft should raise suspicion of infection. Routine cultures and histological examination of the excised corneal buttons are instrumental in the diagnosis of these infections and can guide further treatment.

Transplantation failure due to inadvertent reversal of human donor corneas.

PURPOSE: To emphasize and create awareness of the possibility of inadvertently grafting an inverted corneal button and describe 4 cases that showed this phenomenon on human donor corneas during preservation and transportation from an eye bank. METHODS: Three out of 4 tissues showed inadvertent inversion during transportation and 1 was identified as inverted during preservation in the eye bank. Out of the 3 tissues that were shipped, 2 tissues were transplanted and 1 was identified as inverted prior to transplantation and shipped back to the eye bank. RESULTS: Four tissues showed inversion at different time points. The anatomy was clearly visible with a naked eye that showed the presence of trabecular meshwork on its outside along with some residual choroid. The endothelial cell count was in the range of transplantation without any mortality even after inversion. The inverted corneas showed a mountain-like shape with grooves as compared to the normal cornea. CONCLUSIONS: Previous reports have identified tissue inversion post-transplantation. We describe inadvertent inversion of donor tissues during preservation and transportation from an eye bank. It is recommended to check the presence of corneo-scleral rim, trabecular meshwork, grooves, and residues of choroid if present inside or outside before transplantation to ensure accurate grafting and avoid transplantation failures.

Bubble technique for Descemet membrane endothelial keratoplasty tissue preparation in an eye bank: air or liquid?

PURPOSE: To compare the big-bubble method using air and liquid as medium of separation for Descemet membrane endothelial keratoplasty (DMEK) lenticule preparation in an eye bank. METHODS: Donor corneas (n=20) were immersed in liquid [tissue culture medium (TCM)]. Air and liquid was injected using a 25-gauge needle in the posterior stroma or as near to the stroma-Descemet membrane (DM) phase as possible to create a complete bubble of larger diameter. The endothelial cell density and mortality were checked pre- and postbubble after deflating the tissue. Four pairs of tissues were used to analyse the intracellular tight junctions and three pairs for histological examination and DNA integrity studies, respectively. RESULTS: The yield obtained using air was 80%, whereas that with liquid was 100%. Single injection was required in six cases; twice in two cases; three and four times in one case each with air bubble, whereas seven cases required single injection; twice in two cases; and thrice in just one case with liquid bubble. The average diameter of the final lenticule was 9.12 (±1.71) mm for air bubble and 9.78 (±1.75) mm for liquid bubble with p=0.4362 (no statistical significance). Endothelial cell mortality postbubble preparation was 8.9 (±12.38)% for air and 6.25 (±9.57)% for liquid (p=0.6268). CONCLUSIONS: DM and endothelium could be separated exclusively using air or liquid bubble. However, liquid bubble seems to have certain advantages over air such as the generation of yield, larger diameter and higher maintenance of endothelial cell density and integrity.

DMEK lenticule preparation from donor corneas using a novel 'SubHyS' technique followed by anterior corneal dissection.

PURPOSE: To describe a novel submerged hydro-separation (SubHyS) technique followed by anterior corneal dissection to prepare a Descemet endothelial graft (DEG) for Descemet's membrane endothelial keratoplasty from human donor corneas. METHODS: 30 human donor corneas were immersed in liquid (organ culture (OC) storage medium). Using a 25-gauge needle, approximately 0.3 mL of OC was injected (SubHyS) in the posterior stroma to create a liquid bubble. The bubbled cornea was mounted onto a modified artificial chamber with the epithelial side facing the air. The endothelium was protected with a viscoelastic solution. The anterior cornea was excised with a Barron radial vacuum trephine and the residual peripheral stroma was removed manually using micro-scissors. The DEG was dismounted and washed. The endothelial cell density (ECD) and mortality of the prepared DEG was determined. All the DEGs were preserved in deturgescent medium for 7 days using a cornea claw which was fixed to the sclera. ECD and mortality were checked post preservation. RESULTS: Complete detachment of Descemet's membrane and stroma was achieved in all 30 cases. Bubble burst was observed in five cases (excluded from the study) due to overfilling of the liquid. The average diameter of the excised DEG was 10.96 mm. The average endothelial cell loss post preservation was 27.69%. Histological analysis confirmed elimination of the residual stroma (n=13). CONCLUSIONS: The DEGs can be preserved in a deturgescent medium for up to 7 days. The procedure provides a standardised, pre-validated (quality assured), pre-separated, no-touch, ready-to-use tissue and also reduces the preparation time. Further, the tissues can be trephined as per the surgeon's convenience and can either be rolled or a contact lens could be used for final delivery of the DEG using a surgical glide.

Descemet membrane endothelial keratoplasty tissue preparation from donor corneas using a standardized submerged hydro-separation method.

PURPOSE: To standardize a novel submerged hydro-separation technique for Descemet membrane endothelial keratoplasty (DMEK) graft preparation from donor corneal tissues. DESIGN: Experimental study, laboratory investigation. SETTING: The Veneto Eye Bank Foundation, Venice, Italy. STUDY POPULATION: Fifty-four random human donor corneal tissues unsuitable for transplantation. INTERVENTION: Donor corneas were laid in a sterile basin partially filled with tissue culture medium. A 25 gauge needle with 1 mL mounted syringe was filled with the tissue culture medium. The needle (with bevel up) was bent to 90 degrees and was inserted in the posterior cornea initiating beneath the trabecular meshwork. It was further advanced toward the midperiphery, ensuring that only the bevel was inserted, considering it as a threshold of insertion. The liquid was injected with a medium to high pressure into the posterior stroma or in the Descemet membrane-stroma interface to create the bubble. The tissues were preserved for 7 days in tissue culture medium at 31°C. Parametrical, physiological and histological analyses were carried out. MAIN OUTCOME MEASURES: Larger-diameter tissue, no tissue wastage, reproducibility, and preshipment evaluation. RESULTS: Complete detachment was achieved in all the cases without any tissue wastage. Average diameter of the excised graft was 10.80 (±0.28) mm and endothelial cell loss post preservation was 11.48%. Expression of tight junction protein and regular morphology was observed post preservation. No signs of cell apoptosis were seen. Histological analysis showed elimination of residual stroma in most of the cases. CONCLUSIONS: The submerged hydro-separation method reduces tissue wastage. It allows preshipment evaluation, thus allowing a validated tissue to be transported from the eye banks to the surgeon. Because of the liquid interface, the peeling of the DMEK graft becomes easy for transplantation.

Posterior lamellar graft preparation: a prospective review from an eye bank on current and future aspects.

Descemet membrane endothelial keratoplasty (DMEK) is a corneal surgical technique which selectively replaces the damaged posterior part of the cornea with a healthy donor graft retaining the rest of the tissue intact. There is a need to validate and standardize the donor tissue before grafting due to certain issues that can lead to consequences such as graft failure due to poor endothelial cell count, higher mortality, detachment of the graft, or increased surgical expenses, time, and effort. Thus, prospective potential surgeons and eye banks should now aim at developing new improved surgical techniques in order to prepare the best suited, validated, precut, preloaded, and easy to transplant tissue to reduce pre- and postsurgical complications. This could be achieved by defining parameters like graft thickness, accepted mortality threshold of the endothelial cells, and behavior of grafts during preservation and transportation along with using more sophisticated instruments like microkeratome and femtosecond lasers for graft preparation. Thus, a rapport between the eye banks and the surgeons along with the advanced instruments can overcome this challenge to find the best possible solution for endothelial keratoplasty (EK).

Effect of postmortem interval on the graft endothelium during preservation and after transplantation for keratoconus.

PURPOSE: To investigate the effect of postmortem intervals and prognostic factors on endothelial cell density (ECD) of human donor corneas during preservation and at 1 and 3 years after transplantation in patients transplanted for keratoconus. METHODS: Two different studies were performed: (1) with 733 donor corneas selected for the preservation study and (2) 64 patients with keratoconus selected retrospectively from 2 hospital clinics. The corneas were evaluated on the basis of the ECD during preservation, study A, and at 1 and 3 years after transplantation, study B. The effect of ≥ 10 hours of postmortem interval on the percentage of corneal endothelial cell loss (ECL) was determined. RESULTS: The multiple regression showed no statistical significance (P = 0.827) of postmortem interval on ECL during preservation. However, for patients with keratoconus, the postmortem interval was statistically significant at both 1 year (P < 0.0001) and 3 years after transplantation (P < 0.0001). CONCLUSIONS: The postmortem interval has no influence on the ECD during preservation. However, it has a statistically significant effect on the ECL after transplantation for patients transplanted for keratoconus, and therefore, it becomes eligible to be one of the potential factors affecting the ECD apart from surgical trauma.

A simplified technique for in situ excision of cornea and evisceration of retinal tissue from human ocular globe.

Enucleation is the process of retrieving the ocular globe from a cadaveric donor leaving the rest of the globe undisturbed. Excision refers to the retrieval of ocular tissues, especially cornea, by cutting it separate from the ocular globe. Evisceration is the process of removing the internal organs referred here as retina. The ocular globe consists of the cornea, the sclera, the vitreous body, the lens, the iris, the retina, the choroid, muscles etc (Suppl. Figure 1). When a patient is suffering from corneal damage, the cornea needs to be removed and a healthy one must be transplanted by keratoplastic surgeries. Genetic disorders or defects in retinal function can compromise vision. Human ocular globes can be used for various surgical procedures such as eye banking, transplantation of human cornea or sclera and research on ocular tissues. However, there is little information available on human corneal and retinal excision, probably due to the limited accessibility to human tissues. Most of the studies describing similar procedures are performed on animal models. Research scientists rely on the availability of properly dissected and well-conserved ocular tissues in order to extend the knowledge on human eye development, homeostasis and function. As we receive high amount of ocular globes out of which approximately 40% (Table 1) of them are used for research purposes, we are able to perform huge amount of experiments on these tissues, defining techniques to excise and preserve them regularly. The cornea is an avascular tissue which enables the transmission of light onto the retina and for this purpose should always maintain a good degree of transparency. Within the cornea, the limbus region, which is a reservoir of the stem cells, helps the reconstruction of epithelial cells and restricts the overgrowth of the conjunctiva maintaining corneal transparency and clarity. The size and thickness of the cornea are critical for clear vision, as changes in either of them could lead to distracted, unclear vision. The cornea comprises of 5 layers; a) epithelium, b) Bowman's layer, c) stroma, d) Descemet's membrane and e) endothelium. All layers should function properly to ensure clear vision(4,5,6). The choroid is the intermediate tunic between the sclera and retina, bounded on the interior by the Bruch's membrane and is responsible for blood flow in the eye. The choroid also helps to regulate the temperature and supplies nourishment to the outer layers of the retina(5,6). The retina is a layer of nervous tissue that covers the back of the ocular globe (Suppl. Figure 1) and consists of two parts: a photoreceptive part and a non-receptive part. The retina helps to receive the light from the cornea and lens and converts it into the chemical energy eventually transmitted to the brain with help of the optic nerve(5,6). The aim of this paper is to provide a protocol for the dissection of corneal and retinal tissues from human ocular globes. Avoiding cross-contamination with adjacent tissues and preserving RNA integrity is of fundamental importance as such tissues are indispensable for research purposes aimed at (i) characterizing the transcriptome of the ocular tissues, (ii) isolating stem cells for regenerative medicine projects, and (iii) evaluating histological differences between tissues from normal/affected subjects. In this paper we describe the technique we currently use to remove the cornea, the choroid and retinal tissues from an ocular globe. Here we provide a detailed protocol for the dissection of the human ocular globe and the excision of corneal and retinal tissues. The accompanying video will help researchers to learn an appropriate technique for the retrieval of precious human tissues which are difficult to find regularly.