Mohit Parekh

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. METHODS 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).

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 Descemets 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 Descemets 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.

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

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.

Effect of connexin 43 inhibition by the mimetic peptide Gap27 on corneal wound healing, inflammation and neovascularization

The connexin 43 (Cx43) mimetic peptide Gap27 was designed to transiently block the function of this gap junction. This study was undertaken to investigate the effect of Gap27 on corneal healing, inflammation and neovascularization.

Preloaded donor corneal lenticules in a new validated 3D printed smart storage glide for Descemet stripping automated endothelial keratoplasty

Purpose To design and validate the efficacy of three-dimensional (3D) printed smart storage glide (SSG) which is capable of preserving and delivering posterior lenticules for Descemet stripping automated endothelial keratoplasty (DSAEK). Methods Laboratory investigation (A) was followed by clinical validation (B). Unsuitable corneas for transplantation (n=20) were used for study A. These tissues were cut using a standard microkeratome, punched and loaded into the SSG and preserved for 7 days in transport media. Endothelial cell density (ECD), Trypan blue and Alizarin red staining for endothelial morphology, thickness measurements and glucose uptake, cell apoptosis and immunostaining post preservation were analysed. For study B, clinical grade corneas (n=14) were preloaded in SSG and grafted in patients with indications of Fuchs’ dystrophy (n=8), pseudophakic bullous keratopathy (n=3), posterior polymorphous dystrophy (n=2), and previous DSAEK failure (n=1). Standard DSAEK included descemetorhexis under air and bimanual delivery of the tissue under infusion of buffered saline solution through an anterior chamber maintainer placed at the 12 o’clock position. Main outcome measures for study B were less surgical time, best spectacle-corrected visual acuity (BSCVA), speed of visual recovery, and ECD. Results For study A, an average ECD loss was 2.30±3.21%, thickness increased by 30.80±20.85% and one-third of glucose was utilised during the preservation phase. Alizarin red showed hexagonality of the cells. Cell apoptosis was not observed and expression of ZO-1 was noted on the preserved tissues. In study B, 25% ECD loss was observed after 6 months. BSCVA improved to 20/25 or better within 3 months after DSAEK. Mean surgical time recorded was 21 min. Conclusions This paper describes the development, design, laboratory and clinical validation of a 3D printed SSG which helps to store and deliver posterior lenticules, therefore allowing transportation of quality-controlled precut tissues.

Gene transfer of integration defective anti-HSV-1 meganuclease to human corneas ex vivo.

Corneal graft rejection is a major problem in chronic herpetic keratitis (HK) patients with latent infection. A new class of antiviral agents targeting latent and active forms of herpes simplex virus type 1 (HSV-1) is importantly required. Meganucleases are sequence-specific homing endonucleases capable of inducing DNA double-strand breaks. A proof-of-concept experiment has shown that tailor-made meganucleases are efficient against HSV-1 in vitro. To take this work a step forward, we hypothesized that the pre-treatment of human corneas in eye banks using meganuclease-encoding vectors will allow HK patients to receive a medicated cornea to resist the recurrence of the infection and the common graft rejection problem. However, this strategy requires efficient gene delivery to human corneal endothelium. Using recombinant adeno-associated virus, serotype 2/1 (rAAV2/1), efficient gene delivery of a reporter gene was demonstrated in human corneas ex vivo. The optimum viral dose was 3.7 × 1011 VG with an exposure time of 1 day, followed by 6 days incubation in de-swelling medium. In addition, 12 days incubation can result in transgene expression in excess of 70%. Using similar transduction conditions, meganuclease transgene expression was detected in 39.4% of the endothelial cells after 2 weeks in culture. Reduction of the total viral load in the media and the endothelial cells of corneas infected with HSV-1 was shown. Collectively, this work provides information about the optimum conditions to deliver genetic material to the cornea, and demonstrates for the first time the expression of meganuclease in human corneas ex vivo and its antiviral activity. In conclusion, we demonstrate that the treatment of human corneas in eye banks before transplantation is a new approach to address the unmet clinical needs in corneal diseases.

A Novel De Novo Missense Mutation in TP63 Underlying Germline Mosaicism in AEC Syndrome: Implications for Recurrence Risk and Prenatal Diagnosis

Ankyloblepharon–ectodermal defects–cleft lip/palate (AEC) syndrome is a rare autosomal dominant ectodermal dysplasia syndrome. It is caused by heterozygous mutations in TP63, encoding a transcriptional factor of the p53 family. Mutations in TP63, mainly missense in exons 13 and 14 encoding the sterile alpha motif (SAM) and the transactivation inhibitory (TI) domains, account for 99% of mutations in individuals with AEC syndrome. Of these, ≥70% are de novo mutations, present in the affected patient, but not in parents nor in healthy siblings. However, when a mutation appears de novo, it is not possible to differentiate between a sporadic mutation, or germline mosaicism in the parents. In this latter case, there is a risk of having additional affected offspring. We describe two sisters with AEC syndrome, whose parents were unaffected. Both patients carried the heterozygous c.1568T>C substitution in exon 13 of TP63, resulting in a p.L523P change in the SAM domain of the protein. Analyses of DNA from parental blood cells, seminal fluid (from the father) and maternal cells (buccal, vaginal, and cervical) did not reveal the mutation, suggesting that the mosaicism may involve a very low percentage of cells (very low grade somatic mosaicism) or, more likely, maternal gonadal mosaicism. Mosaicism must be considered for the assessment of recurrence risk during genetic counseling in AEC syndrome, and pre‐implantation/prenatal genetic diagnosis should be offered to all couples, even when the mutation is apparently de novo.

Endothelium-in versus endothelium-out for Descemet membrane endothelial keratoplasty graft preparation and implantation.

To evaluate the difference between endothelium‐in and endothelium‐out for Descemet membrane endothelial keratoplasty (DMEK) preparation and implantation using injection method.

Concise Review: An Update on the Culture of Human Corneal Endothelial Cells for Transplantation

The cornea forms the front window of the eye, enabling the transmission of light to the retina through a crystalline lens. Many disorders of the cornea lead to partial or total blindness, and therefore corneal transplantation becomes mandatory. Recently, selective corneal layer (as opposed to full thickness) transplantation has become popular because this leads to earlier rehabilitation and visual outcomes. Corneal endothelial disorders are a common cause of corneal disease and transplantation. Corneal endothelial transplantation is successful but limited worldwide because of lower donor corneal supply. Alternatives to corneal tissue for endothelial transplantation therefore require immediate attention. The field of human corneal endothelial culture for transplantation is rapidly emerging as a possible viable option. This manuscript provides an update regarding these developments.