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Here we describe a new, non-human, ex-vivo model (goat eye model) for training surgeons in DMEK surgeons. In a wet lab setting, goat eyes were used to obtain a pseudo-DMEK graft of 8 mm from the goat lens capsule that was injected into another goat eye with the same maneuvers described for human DMEK. The DMEK pseudo-graft can be easily prepared, stained, loaded, injected, and unfolded into the goat eye model reproducing the similar maneuvers used for DMEK in a human eye, except for the descemetorhexis, which cannot be performed. The pseudo-DMEK graft behaves similar to human DMEK graft and useful for surgeons to experience and understand steps of DMEK early in learning curve. The concept of a non-human ex-vivo eye model is simple and reproducible and obviates the need for human tissue and the issues of poor visibility in stored corneal tissue.

Following corneal transplantation, there is an initial, rapid decline in corneal endothelial cells (CECs) following surgery. Direct imaging of post-transplantation endothelial cells is only possible weeks after surgery and with a limited field of view. We have developed a labelling approach using 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide (DIR) dye solution, that enables tracking of labelled CECs in vivo for at least 1 month. Initial in vitro optimization, with assessments of dye concentration on fluorescence, cellular toxicity and cell migration, performed in propagated primary CECs. Subsequently, in vivo evaluation of cellular labelling was assessed within a rabbit wound healing model. Finally, real-time visualization of human cadaver donor tissue incubated in DIR transplanted into rabbits was achieved using a clinical confocal microscope. Results revealed detectable fluorescence increased with concentration to a plateau of 100 µg/ml, with no toxicity of CECs at any concentration evaluated. DIR-labelled CECs were detectable in vivo up to 1 month, and transplanted labelled donor graft could be visualized and were trackable in vivo. Acute endothelial rejection in 1 rabbit was evidenced by detectable DIR positive cells within the anterior chamber. DIR imaging allowed for detailed imaging of the transplanted human corneal endothelium, and enabled non-invasive observation of the corneal endothelial morphology following transplantation.

To establish a method for assessing graft viability, in-vivo, following corneal transplantation. Optimization of calcein AM fluorescence and toxicity assessment was performed in cultured human corneal endothelial cells and ex-vivo corneal tissue. Descemet membrane endothelial keratoplasty grafts were incubated with calcein AM and imaged pre and post preparation, and in-situ after insertion and unfolding in a pig eye model. Global, macroscopic images of the entire graft and individual cell resolution could be attained by altering the magnification of a clinical confocal scanning laser microscope. Patterns of cell loss observed in situ were compared to those seen using standard ex-vivo techniques. Calcein AM showed a positive dose-fluorescence relationship. A dose of 2.67μmol was sufficient to allow clear discrimination between viable and non-viable areas (sensitivity of 96.6% with a specificity of 96.1%) and was not toxic to cultured endothelial cells or ex-vivo corneal tissue. Patterns of cell loss seen in-situ closely matched those seen on ex-vivo assessment with fluorescence viability imaging, trypan blue/alizarin red staining or scanning electron microscopy. Iatrogenic graft damage from preparation and insertion varied between 7-35% and incarceration of the graft tissue within surgical wounds was identified as a significant cause of endothelial damage. In-situ graft viability assessment using clinical imaging devices provides comparable information to ex-vivo methods. This method shows high sensitivity and specificity, is non-toxic and can be used to evaluate immediate cell viability in new grafting techniques in-vivo.

*Correspondence - Luis García-Onrubia: luis.garciaonrubia@gstt.nhs.ukPurposeTo describe a novel technique for deep anterior lamellar keratoplasty (DALK) in patients central corneal perforation and deep scarring making conventional DALK (Melles or Big Bubble) unviable. A posterior Descemet’s membrane (DM) skirt has provided an adequate scaffold for the migration of the host endothelial cells.Methods and AnalysisA case report. A 32-year-old male with previous hydrops developed severe corneal scarring with a break in DM visible on OCT scanning. A modified DALK procedure was perform consisting of a 400µm, 8.5mm Anterior lamellar cap with a 4.5mm posterior lamellar disc, denuded of endothelial cells and containing a DM skirt.Initially, manual dissection of the and anterior 400µm of corneal stroma was performed using a modified Melles technique. The residual posterior lamellar was assessed and found to have significant residual scarring. A central 4mm optical window was performed through the posterior lamellar over the visual axis.The donor tissue was cut using a 350µm microkeratome head. The anterior cap was trephined to 8.5mm and set aside. The posterior lamellar was placed in a punch block, and the endothelial was removed using a silicone tipped cannula. The removal of endothelial cells was confirmed using trypan blue dye. A posterior lamellar graft with a 4.0mm stromal bed and a 4.5mm DM skirt was fashion using a peeling and double punch technique. The posterior lamellar graft was inserted into the optical window such that the DM skirt provided a bridge to the donor corneal endothelium. The anterior cap was sutured with a double continuous suture of 10–0 monofilament nylon. An inferior peripheral iridotomy was created, and an air bubble filling the anterior chamber was left at the end of the case.ResultsThe preoperative visual acuity (VA) was hand movements. Full attachment of the posterior lamellar was seen at all time-points from week one onwards. Central corneal pachymetry continued to reduce for 12 weeks. One year after the operation, with sutures in, the best spectacle-corrected VA was 6/12. The corneal graft was clear, and no rejection episodes occurred. Endothelial cell repopulation of the donor DM could be observed with specular microscopy.ConclusionThe presence of DM promotes endothelial migration and healing. Modifications to traditional DALK surgery, in which DM is used to promote endothelial healing, are a viable alternative to penetrating keratoplasty. This method eliminates the risk of allograft endothelial rejection and allows a ‘regenerative’ for DALK to be used, offering a new modality of treatment in patients with healthy reserves of endothelial cells and deep posterior lamellar scarring.

*Correspondence - Luis García-Onrubia: luis.garciaonrubia@gstt.nhs.ukObjectiveTo report the clinical outcomes of a series of cases in which localised areas of endothelial function were selectively treated with shape and position matched endothelial transplanted in a procedure we have termed Descemet’s membrane endothelial patching (DMEP).MethodsInterventional case series. Five patients presented with localised endothelial dysfunction in eyes with high-risk graft failure due either to rejection, recurrence of the focal endothelial dysfunction or because extended treatment with steroid drops was contraindicated. Endothelial grafts matching the area of dysfunction were produced to preserve healthy host cells and limit the immunological burden of new grafts. Patient demographic details, indication for surgery, preoperative and postoperative visual acuity, intraoperative and postoperative complications, and graft rejections episodes were noted.ResultsFive patients were included in this cases series. Indications for DMEP were Fuchs’ heterochromic iridocyclitis (n=1), Fuchs’ endothelial dystrophy (n=2), endotheliitis (n=2). In all cases, a customised DMEP graft was used, as opposed to our standard 8.25mm circular DMEK graft size. The DMEP grafts were centred over the area of focal endothelial dysfunction. In all cases, complete graft attachment was achieved, and the corneas were cleared. Steroid drops were reduced rapidly without any episodes of graft rejection/failure reported at 1 year.ConclusionDMEP transplants are a viable option to treat localised endothelial dysfunction. Placing non-circular, no central transplants is surgically feasible and does not appear to affect graft adhesion. Limiting the size of the transplant may limit the immunological burden of new grafts and reduce the need for extended courses of steroids.

Donor corneas with low endothelial cell densities (ECD) are deemed unsuitable for corneal endothelial transplantation. This study evaluated a two-step incubation and dissociation harvesting approach to isolate single corneal endothelial cells (CECs) from donor corneas for corneal endothelial cell-injection (CE-CI) therapy. To isolate CECs directly from donor corneas, optimization studies were performed where donor Descemet’s membrane/corneal endothelium (DM/CE) were peeled and incubated in either M4-F99 or M5-Endo media before enzymatic digestion. Morphometric analyses were performed on the isolated single cells. The functional capacities of these cells, isolated using the optimized simple non-cultured endothelial cells (SNEC) harvesting technique, for CE-CI therapy were investigated using a rabbit bullous keratopathy model. The two control groups were the positive controls, where rabbits received cultured CECs, and the negative controls, where rabbits received no CECs. Whilst it took longer for CECs to dislodge as single cells following donor DM/CE incubation in M5-Endo medium, CECs harvested were morphologically more homogenous and smaller compared to CECs obtained from DM/CE incubated in M4-F99 medium (p < 0.05). M5-Endo medium was hence selected as the DM/CE incubation medium prior to enzymatic digestion to harvest CECs for the in vivo cell-injection studies. Following SNEC injection, mean central corneal thickness (CCT) of rabbits increased to 802.9 ± 147.8 μm on day 1, gradually thinned, and remained clear with a CCT of 385.5 ± 38.6 μm at week 3. Recovery of corneas was comparable to rabbits receiving cultured CE-CI (p = 0.40, p = 0.17, and p = 0.08 at weeks 1, 2, and 3, respectively). Corneas that did not receive any cells remained significantly thicker compared to both SNEC injection and cultured CE-CI groups (p < 0.05). This study concluded that direct harvesting of single CECs from donor corneas for SNEC injection allows the utilization of donor corneas unsuitable for conventional endothelial transplantation.

Arterialised blood flow within the cavernous sinus causes an ocular bruit, pulsating exophthalmos, diplopia, conjunctival chemosis, orbital pain, dilated and arterialised conjunctival vessels, raised intraocular pressure, and proptosis. Recanalisation of the occluded brachiocephalic vein was an alternative therapeutic option but we chose not to do this because of the long-standing nature of the occlusion, and intimal hyperplasia caused by the shunt that would have increased the likelihood of stent occlusion.

PurposeTo describe a novel method of global cell viability assessment for Descemet membrane endothelial keratoplasty (DMEK) and the comparison of two contemporary methods of donor tissue preparation.MethodsDMEK transplants were prepared using two different methods: liquid bubble separation and manual peeling (n=8 each group). Samples were incubated with Hoechst, calcein-AM and ethidium homodimer prior to mounting on a curved imaging chamber. Z-stacked fluorescence microscopy images were combined to produce an in-focus global image capable of resolving all cell nuclei. Image processing software was used to define a calcein-positive live cell area, count all cell nuclei within this area and subtract ethidium-positive dead cells to derive the total viable endothelial cell count. Corrected global cell density was calculated by dividing the number of viable cells by the graft area, which had been corrected for imaging a curved surface.ResultsCorrected global cell density was lower than the central endothelial cell density in both groups: 85.5% of the pre-preparation central endothelial cell density in the peel group and 75.8% in the bubble group. Corrected global cell density was significantly lower in the liquid bubble separation group than in the peel group (p=0.04).ConclusionsEye bank estimations of central endothelial cell density overestimate true cell density after graft preparation in DMEK. A peel method is less damaging and more consistent than a liquid bubble method. Cell loss correlated strongly with the degree of stromal hydration prior to bubble separation in the liquid bubble group.

Here we describe a new, non-human, ex-vivo model (goat eye model) for training surgeons in DMEK surgeons. In a wet lab setting, goat eyes were used to obtain a pseudo-DMEK graft of 8 mm from the goat lens capsule that was injected into another goat eye with the same maneuvers described for human DMEK. The DMEK pseudo-graft can be easily prepared, stained, loaded, injected, and unfolded into the goat eye model reproducing the similar maneuvers used for DMEK in a human eye, except for the descemetorhexis, which cannot be performed. The pseudo-DMEK graft behaves similar to human DMEK graft and useful for surgeons to experience and understand steps of DMEK early in learning curve. The concept of a non-human ex-vivo eye model is simple and reproducible and obviates the need for human tissue and the issues of poor visibility in stored corneal tissue.

Corneal transplantation is the only treatment available to restore vision for individuals with blindness due to corneal endothelial dysfunction. However, severe shortage of available donor corneas remains a global challenge. Functional regulatory compliant tissue-engineered corneal endothelial graft substitute can alleviate this reliance on cadaveric corneal graft material. Here, isolated primary human corneal endothelial cells (CEnCs) propagated using a dual media approach refined towards regulatory compliance showed expression of markers indicative of the human corneal endothelium, and can be tissue-engineered onto thin corneal stromal carriers. Both cellular function and clinical adaptability was demonstrated in a pre-clinical rabbit model of bullous keratopathy using a tissue-engineered endothelial keratoplasty (TE-EK) approach, adapted from routine endothelial keratoplasty procedure for corneal transplantation in human patients. Cornea thickness of rabbits receiving TE-EK graft gradually reduced over the first two weeks, and completely recovered to a thickness of approximately 400 µm by the third week of transplantation, whereas corneas of control rabbits remained significantly thicker over 1,000 µm ( p  < 0.05) throughout the course of the study. This study showed convincing evidence of the adaptability of the propagated CEnCs and their functionality via a TE-EK approach, which holds great promises in translating the use of cultured CEnCs into the clinic.