Graft-host Interface Research Articles

Post-DSAEK Optical Changes

The aim was to assess the visual impact of ocular wavefront aberrations, corneal thickness, and corneal light scatter prospectively after performing a Descemet stripping automated endothelial keratoplasty (DSAEK) in humans. Data were obtained prospectively from 20 eyes preoperatively and at 1, 3, 6, and 12 months post-DSAEK. At each visit, the best spectacle-corrected visual acuity and visual acuity with glare (brightness acuity testing) were recorded, and ocular wavefront measurements and corneal optical coherence tomography (OCT) were performed. The magnitude and the sign of individual Zernike terms [higher-order aberrations (HOAs)] were determined. Epithelial, host stromal, donor stromal, and total corneal thicknesses were quantified. The brightness and intensity profiles of OCT images were generated to quantify light scatter in the whole cornea, subepithelial region, anterior and posterior host stroma, interface, and donor stroma. The mean best spectacle-corrected visual acuity and glare disability at low light levels improved from 1 to 12 months post-DSAEK. All corneal thicknesses and ocular lower-order aberrations and HOAs were found to be stable from 1 to 12 months, whereas total corneal, host stromal, and interface brightness intensities decreased significantly over the same period. A repeated measures analysis of variance performed across the follow-up period revealed that the change in scatter, but not the change in the HOAs, could account for the variability occurring in the acuity from 1 to 12 months post-DSAEK. Although ocular HOAs and scatter are both elevated over normal values post-DSAEK, our results demonstrate that the improvements in visual performance occurring over the first year post-DSAEK are associated with decreasing light scatter. In contrast, there were no significant changes in the ocular HOAs during this time. Because corneal light scatter decreased between 1 and 12 months despite there being stable corneal thicknesses over the same period, we conclude that factors that induced light scatter, other than tissue thickness or swelling (corneal edema), significantly impacted the visual improvements that occurred over time post-DSAEK. A better understanding of the cellular and extracellular matrix changes of the subepithelial region and interface, incurred by the surgical creation of a lamellar host-graft interface, and the subsequent healing of these tissues, is warranted.

Inadvertent Placement of a Glaucoma Tube Into the Graft-Host Interface of an Eye With Previous Descemet Stripping Automated Endothelial Keratoplasty

The aim of this study was to report the inadvertent insertion of a glaucoma tube into the graft-host interface in a patient with previous Descemet stripping automated endothelial keratoplasty (DSAEK). This is a retrospective case report. A 56-year-old aniridic woman with a history of multiple failed penetrating keratoplasties (PKPs) and glaucoma underwent DSAEK under failed PKP of her left eye at our institution. After undergoing a subsequent glaucoma tube placement by an outside glaucoma specialist, the tube was identified in the DSAEK-PKP interface. The tube was successfully repositioned at the time of repeat PKP with placement of a Boston type I keratoprosthesis. Inadvertent insertion of a glaucoma tube into the DSAEK interface may result in ocular morbidity in patients with previous DSAEK surgery.

Keratocyte Apoptosis and Not Myofibroblast Differentiation Mark the Graft/Host Interface at Early Time-Points Post-DSAEK in a Cat Model

PurposeTo evaluate myofibroblast differentiation as an etiology of haze at the graft-host interface in a cat model of Descemet’s Stripping Automated Endothelial Keratoplasty (DSAEK).MethodsDSAEK was performed on 10 eyes of 5 adult domestic short-hair cats. In vivo corneal imaging with slit lamp, confocal, and optical coherence tomography (OCT) were performed twice weekly. Cats were sacrificed and corneas harvested 4 hours, and 2, 4, 6, and 9 days post-DSAEK. Corneal sections were stained with the TUNEL method and immunohistochemistry was performed for α-smooth muscle actin (α-SMA) and fibronectin with DAPI counterstain.ResultsAt all in vivo imaging time-points, corneal OCT revealed an increase in backscatter of light and confocal imaging revealed an acellular zone at the graft-host interface. At all post-mortem time-points, immunohistochemistry revealed a complete absence of α-SMA staining at the graft-host interface. At 4 hours, extracellular fibronectin staining was identified along the graft-host interface and both fibronectin and TUNEL assay were positive within adjacent cells extending into the host stroma. By day 2, fibronectin and TUNEL staining diminished and a distinct acellular zone was present in the region of previously TUNEL-positive cells.ConclusionsOCT imaging consistently showed increased reflectivity at the graft-host interface in cat corneas in the days post-DSAEK. This was not associated with myofibroblast differentiation at the graft-host interface, but rather with apoptosis and the development of a subsequent acellular zone. The roles of extracellular matrix changes and keratocyte cell death and repopulation should be investigated further as potential contributors to the interface optical changes.

Correction: Quantification of Collagen Ultrastructure after Penetrating Keratoplasty – Implications for Corneal Biomechanics

[This corrects the article on p. e68166 in vol. 8.].

Bone cysts after osteochondral allograft repair of cartilage defects in goats suggest abnormal interaction between subchondral bone and overlying synovial joint tissues

The efficacy of osteochondral allografts (OCAs) may be affected by osseous support of the articular cartilage, and thus affected by bone healing and remodeling in the OCA and surrounding host. Bone cysts, and their communication pathways, may be present in various locations after OCA insertion and reflect distinct pathogenic mechanisms. Previously, we analyzed the effect of OCA storage (FRESH, 4°C/14d, 4°C/28d, FROZEN) on cartilage quality in fifteen adult goats after 12months in vivo. The objectives of this study were to further analyze OCAs and contralateral non-operated (Non-Op) CONTROLS from the medial femoral condyle to (1) determine the effect of OCA storage on local subchondral bone (ScB) and trabecular bone (TB) structure, (2) characterize the location and structure of bone cysts and channels, and (3) assess the relationship between cartilage and bone properties. (1) Overall bone structure after OCAs was altered compared to Non-Op, with OCA samples displaying bone cysts, ScB channels, and ScB roughening. ScB BV/TV in FROZEN OCAs was lower than Non-Op and other OCAs. TB BV/TV in FRESH, 4°C/14d, and 4°C/28d OCAs did not vary compared to Non-Op, but BS/TV was lower. (2) OCAs contained “basal” cysts, localized to deeper regions, some “subchondral” cysts, localized near the bone–cartilage interface, and some ScB channels. TB surrounding basal cysts exhibited higher BV/TV than Non-Op. (3) Basal cysts occurred (a) in isolation, (b) with subchondral cysts and ScB channels, (c) with ScB channels, or (d) with subchondral cysts, ScB channels, and ScB erosion. Deterioration of cartilage gross morphology was strongly associated with abnormal μCT bone structure. Evidence of cartilage–bone communication following OCA repair may favor fluid intrusion as a mechanism for subchondral cyst formation, while bone resorption at the graft–host interface without affecting overall bone and cartilage structure may favor bony contusion mechanism for basal cyst formation. These findings suggest that cysts occurring after OCAs may result from aberrant mechanobiology due to (1) altered compartmentalization that normally separates overlying cartilage and subchondral bone, either from distinct ScB channels or more general ScB plate deterioration, and (2) bone resorption at the basal graft–host interface.

Topical Application of Soluble CD83 Induces IDO-Mediated Immune Modulation, Increases Foxp3+ T Cells, and Prolongs Allogeneic Corneal Graft Survival

Modulation of immune responses is one of the main research aims in transplant immunology. In this study, we investigate the local immunomodulatory properties of soluble CD83 (sCD83) at the graft-host interface using the high-risk corneal transplantation model. In this model, which mimics the inflammatory status and the preexisting vascularization of high-risk patients undergoing corneal transplantation, allogeneic donor corneas are transplanted onto sCD83-treated recipient animals. This model allows the direct and precise application of the immune modulator at the transplantation side. Interestingly, sCD83 was able to prolong graft survival after systemic application as well as after topical application, which is therapeutically more relevant. The therapeutic effect was accompanied by an increase in the frequency of regulatory T cells and was mediated by the immune-regulatory enzyme IDO and TGF-β. In vitro, sCD83 induced long-term IDO expression in both conventional and plasmacytoid dendritic cells via autocrine or paracrine production of TGF-β, a cytokine previously shown to be an essential mediator of IDO-dependent, long-term tolerance. These findings open new treatment avenues for local immune modulation after organ and tissue transplantation.

Quantification of Collagen Ultrastructure after Penetrating Keratoplasty – Implications for Corneal Biomechanics

PurposeTo quantify long-term changes in stromal collagen ultrastructure following penetrating keratoplasty (PK), and evaluate their possible implications for corneal biomechanics.MethodsA pair of 16 mm post-mortem corneo-scleral buttons was obtained from a patient receiving bilateral penetrating keratoplasty 12 (left)/28 (right) years previously. Small-angle x-ray scattering quantified collagen fibril spacing, diameter and spatial order at 0.5 mm or 0.25 mm intervals along linear scans across the graft margin. Corresponding control data was collected from two corneo-scleral buttons with no history of refractive surgery. Wide-angle x-ray scattering quantified collagen fibril orientation at 0.25 mm (horizontal)×0.25 mm (vertical) intervals across both PK specimens. Quantification of orientation changes in the graft margin were verified by equivalent analysis of data from a 13 year post-operative right PK specimen obtained from a second patient in a previous study, and comparison made with new and published data from normal corneas.ResultsMarked changes to normal fibril alignment, in favour of tangentially oriented collagen, were observed around the entire graft margin in all PK specimens. The total number of meridional fibrils in the wound margin was observed to decrease by up to 40%, with the number of tangentially oriented fibrils increasing by up to 46%. As a result, in some locations the number of fibrils aligned parallel to the wound outnumbered those spanning it by up to five times. Localised increases in fibril spacing and diameter, with an accompanying reduction in matrix order, were also evident.ConclusionsAbnormal collagen fibril size and spatial order within the PK graft margin are indicative of incomplete stromal wound remodelling and the long term persistence of fibrotic scar tissue. Lasting changes in collagen fibril orientation in and around PK wounds may alter corneal biomechanics and compromise the integrity of the graft-host interface in the long term.

Histological and ultrastructural findings of corneal tissue after failed descemet membrane endothelial keratoplasty

To investigate in a retrospective review the histological and ultrastructural findings after failed primary and early Descemet membrane endothelial keratoplasty (DMEK), propose possible pathomechanisms of graft failure and give clinical implications. The explanted grafts underwent light- and electromicroscopical investigations in eight failed DMEK cases. Haematoxylin - Eosin, periodic acid Schiff and Alcian blue stainings were performed. Special note was given to any residual stromal remnants, absence of endothelial cells, lamellar structure and 'activation' of keratocytes. Of the eight cases, six were re-DMEKs and two penetrating keratoplasties. Partial graft separation was seen in six and no graft separation in two of the cases. The average time-interval to the re-DMEK or penetrating keratoplasty was 4.6 months. Light and electron microscopy of the two explanted stromal specimens showed varying degrees of keratocyte activation. Endothelial cell loss was observed in essentially all explants with varying degrees and positive correlation with intraoperative difficulty. Assumed upside-down situations showed large areas of intact endothelial cells. In addition, a new layer, situated between the endothelial cell layer and the posterior nonbanded layer, was observed with loose intercellular structure. A loss of the endothelial cell layer of varying degrees and positive correlation with intraoperative difficulty are the prominent feature of primary and early DMEK graft failure. Of note is the upside-down situation, in which in some cases, the endothelial cell layer not only remains intact but also demonstrates metabolical activity in forming a novel cellular layer.

Combined treatment with platelet-rich plasma and brain-derived neurotrophic factor-overexpressing bone marrow stromal cells supports axonal remyelination in a rat spinal cord hemi-section model

Background aimsCombining biologic matrices is becoming a better choice to advance stem cell-based therapies. Platelet-rich plasma (PRP) is a biologic product of concentrated platelets and has been used to promote regeneration of peripheral nerves after injury. We examined whether PRP could induce rat bone marrow stromal cells (BMSCs) differentiation in vitro and whether a combination of BMSCs, PRP and brain-derived neurotrophic factor (BDNF) could provide additive therapeutic benefits in vivo after spinal cord injury (SCI). MethodsBMSCs and BDNF-secreting BMSCs (BDNF-BMSCs) were cultured with PRP for 7 days and 21 days, respectively, and neurofilament (NF)-200, glial fibrillary acidic protein (GFAP), microtubule-associated protein 2 (MAP2) and ribosomal protein S6 kinase (p70S6K) gene levels were assessed. After T10 hemi-section in 102 rats, 15-μL scaffolds (PRP alone, BMSCs, PRP/BMSCs, BDNF-BMSCs or PRP/BDNF-BMSCs) were transplanted into the lesion area, and real-time polymerase chain reaction, Western blot, immunohistochemistry and ultrastructural studies were performed. ResultsThe messenger RNA expression of NF-200, GFAP, MAP2 and p70S6K was promoted in BMSCs and BDNF-BMSCs after culture with PRP in vitro. BDNF levels were significantly higher in the injured spinal cord after implantation of BDNF-BMSCs. In the PRP/BDNF-BMSCs group at 8 weeks postoperatively, more GFAP was observed, with less accumulation of astrocytes at the graft-host interface. Rats that received PRP and BDNF-BMSC implants showed enhanced hind limb locomotor performance at 8 weeks postoperatively compared with control animals, with more axonal remyelination. ConclusionsA combined treatment comprising PRP and BDNF-overexpressing BMSCs produced beneficial effects in rats with regard to functional recovery after SCI through enhancing migration of astrocytes into the transplants and axonal remyelination.

Textural Interface Opacity After Descemet Stripping Automated Endothelial Keratoplasty

Descemet stripping automated endothelial keratoplasty (DSAEK) has its own set of complications including interface abnormalities. This case series presents the largest number of patients who developed textural interface opacity (TIO) at the graft-host interface after DSAEK. This is a retrospective multicenter case series of 30 patients from 7 institutions with the finding of TIO. Clinical information collected included donor preparation details, recipient information, and surgical technique. Clinical outcomes included best-corrected visual acuity and status of TIO appearance at the last follow-up visit. Slit-lamp photographs were analyzed and compared. The majority of the patients (73%) had a best-corrected visual acuity of 20/40 or better. Four of the donor tissues were prepared with a microkeratome blade with the same lot number. Six patients had a central interface space between host and donor stromal surfaces--presumed interface fluid but potentially viscoelastic. A slight majority (57%) of patients had improvement in the severity of TIO, with 20% noted to have a complete resolution of TIO (mean follow-up of 11.9 months). Two clinical types of TIO were seen: an elongated type and a punctate type. Most patients with TIO after DSAEK obtain good visual outcomes. TIO spontaneously improves or even resolves during follow-up without intervention. The etiology of this condition is unknown, but we propose 2 different mechanisms. The elongated type could be secondary to an irregular cut of the donor with the microkeratome blade. The punctate type may be secondary to retained viscoelastic.

Invasion néovasculaire de l’interface endothélio-descemetique au décours d’une kératoplastie lamellaire antérieure profonde

The endothelio-descemetic interface (EDI) of a deep anterior lamellar keratoplasty (DALK) may become the locus of a neovascular proliferation. Bevacizumab (Avastin) is an antiangiogenic alternative available for curative treatment in deep corneal neovascularization. We report the case of a 49-year-old woman who had received two arcuate relaxing incisions for high astigmatism after DALK. She developed graft rejection associated with significant neovascular stromal invasion in the EDI of the graft-host interface. Two subconjunctival injections of bevacizumab 1 month apart as well as thermocauterization of the feeding vessel were necessary and effective. No recurrence has been observed after 2 years of follow-up. Corneal neovascularization can be a cause of DALK rejection. A combination of anti-antiangiogenic and physical methods may abort graft rejection and permanently restore graft function.

Implications of poly(N-isopropylacrylamide)-g-poly(ethylene glycol) with codissolved brain-derived neurotrophic factor injectable scaffold on motor function recovery rate following cervical dorsolateral funiculotomy in the rat

In a follow-up study to their prior work, the authors evaluated a novel delivery system for a previously established treatment for spinal cord injury (SCI), based on a poly(N-isopropylacrylamide) (PNIPAAm), lightly cross-linked with a polyethylene glycol (PEG) injectable scaffold. The primary aim of this work was to assess the recovery of both spontaneous and skilled forelimb function following a cervical dorsolateral funiculotomy in the rat. This injury ablates the rubrospinal tract (RST) but spares the dorsal and ventral corticospinal tract and can severely impair reaching and grasping abilities. Animals received an implant of either PNIPAAm-g-PEG or PNIPAAm-g-PEG + brain-derived neurotrophic factor (BDNF). The single-pellet reach-to-grasp task and the staircase-reaching task were used to assess skilled motor function associated with reaching and grasping abilities, and the cylinder task was used to assess spontaneous motor function, both before and after injury. Because BDNF can stimulate regenerating RST axons, the authors showed that animals receiving an implant of PNIPAAm-g-PEG with codissolved BDNF had an increased recovery rate of fine motor function when compared with a control group (PNIPAAm-g-PEG only) on both a staircase-reaching task at 4 and 8 weeks post-SCI and on a single-pellet reach-to-grasp task at 5 weeks post-SCI. In addition, spontaneous motor function, as measured in the cylinder test, recovered to preinjury values in animals receiving PNIPAAm-g-PEG + BDNF. Fluorescence immunochemistry indicated the presence of both regenerating axons and BDA-labeled fibers growing up to or within the host-graft interface in animals receiving PNIPAAm-g-PEG + BDNF. Based on their results, the authors suggest that BDNF delivered by the scaffold promoted the growth of RST axons into the lesion, which may have contributed in part to the increased recovery rate.

A Study of Host Corneal Endothelial Cells After Non-Descemet Stripping Automated Endothelial Keratoplasty

To determine the short-term fate of the host endothelium and Descemet membrane after non-Descemet stripping automated endothelial keratoplasty (nDSAEK). Eight unilateral DSAEK (n = 4) or nDSAEK (n = 4) surgeries were performed in the right eyes of 8 rabbits. Corneal transparency and thickness were followed-up by slit-lamp microscopy, and 2 weeks postoperatively, corneas were evaluated by immunohistochemistry and transmission electron microscopy. Corneas remained clear after both DSAEK and nDSAEK. One week after DSAEK, the stroma-to-stroma surgical interface was identifiable as a zone of fibrotic tissue a few microns thick, whereas in the nDSAEK group, the recipient corneal endothelium and Descemet membrane were clearly visible at the graft-host interface. The retained endothelial cells were positive for Na/K-ATPase but assumed a markedly different morphology from healthy endothelial cells, with cell processes extending into the graft stroma or engulfing strands of irregularly dissected grafted stromal tissue where they occasionally appeared to compartmentalize the transplanted matrix and became detached from the underlying Descemet membrane. Host endothelial cells 2 weeks after nDSAEK express markers of pump function, but appear to be morphologically altered, occasionally detaching from the adjacent Descemet membrane, extending into the graft stroma or engulfing strands of the grafted stroma at the interface. The short-term persistence and subsequent phenotypical alternation of residual endothelial cells, aligned to structural changes to Descemet membrane, might influence graft adherence after nDSAEK.

Integration and Long Distance Axonal Regeneration in the Central Nervous System from Transplanted Primitive Neural Stem Cells

Spinal cord injury (SCI) results in devastating motor and sensory deficits secondary to disrupted neuronal circuits and poor regenerative potential. Efforts to promote regeneration through cell extrinsic and intrinsic manipulations have met with limited success. Stem cells represent an as yet unrealized therapy in SCI. Recently, we identified novel culture methods to induce and maintain primitive neural stem cells (pNSCs) from human embryonic stem cells. We tested whether transplanted human pNSCs can integrate into the CNS of the developing chick neural tube and injured adult rat spinal cord. Following injection of pNSCs into the developing chick CNS, pNSCs integrated into the dorsal aspects of the neural tube, forming cell clusters that spontaneously differentiated into neurons. Furthermore, following transplantation of pNSCs into the lesioned rat spinal cord, grafted pNSCs survived, differentiated into neurons, and extended long distance axons through the scar tissue at the graft-host interface and into the host spinal cord to form terminal-like structures near host spinal neurons. Together, these findings suggest that pNSCs derived from human embryonic stem cells differentiate into neuronal cell types with the potential to extend axons that associate with circuits of the CNS and, more importantly, provide new insights into CNS integration and axonal regeneration, offering hope for repair in SCI.

A Cause of Reticular Interface Haze and its Management After Descemet Stripping Endothelial Keratoplasty

To describe a cause and management of interface haze after Descemet stripping endothelial keratoplasty (DSEK). Five patients underwent uncomplicated combined DSEK/phacoemulsification with intraocular lens implantation at a tertiary referral center over a span of 3 months. In each case, the recipient Descemet membrane was stripped with the anterior chamber filled with a cohesive viscoelastic. Patients subsequently developed visually significant haze in the graft-host interface that had a fine reticular pattern. Examination also revealed a small separation between the donor and recipient cornea in 3 cases. In all cases, there was no communication between the area of detachment and the anterior chamber, and the overlying recipient cornea was clear. Clinical and imaging findings of the interface haze suggested that it was the result of retained viscoelastic in the interface. Patients were either observed or underwent surgery to irrigate the viscoelastic from the interface. Of the 5 patients, 3 were simply observed with eventual clearing of the haze and attainment of best-corrected visual acuity of 20/40 or better. Two patients with more extensive interface haze and visible space on slit examination between the donor and recipient cornea underwent irrigation and aspiration of the graft-host interface, with quick resolution of the haze. All grafts remained clear at the last follow-up. Retained viscoelastic at the graft-host interface during DSEK can cause a reticular interface haze that can compromise visual clarity. The haze can be eliminated by irrigating and aspirating the graft-host interface with improvement in vision. Careful irrigation and aspiration of viscoelastic from the posterior corneal surface of the recipient at the time of endothelial keratoplasty can prevent this form of interface haze.

Autogenous Impaction Grafting in Total Hip Arthroplasty with Developmental Dysplasia of the Hip

Autogenous impaction grafting was performed to improve acetabular cup coverage in 68 patients (78 hips) with developmental dysplasia of the hip (DDH) after total hip arthroplasty, and the patients were then monitored for a mean duration of 5.5years. No cup revisions were required, and there was no radiographic loosening. The host–graft interface became indistinct by 6weeks after surgery, trabecular bone formation was seen by 12weeks, and definite trabecular reorientation was seen by 20months. At the last follow-up examination, the mean cup coverage was 97.7%, whereas mean coverage by bone grafting was 27.9%. Among 12 patients with most of the bone graft placed at the absolutely protruded area of the cup, the cup coverage provided by the bone graft decreased from 95.4% immediately after surgery to 20.7% by the final follow-up examination. Bone-particle grafting can provide a satisfactory integration speed and a low incidence of displacement and ectopic ossification.

Exogenous BDNF enhances the integration of chronically injured axons that regenerate through a peripheral nerve grafted into a chondroitinase-treated spinal cord injury site

Although axons lose some of their intrinsic capacity for growth after their developmental period, some axons retain the potential for regrowth after injury. When provided with a growth-promoting substrate such as a peripheral nerve graft (PNG), severed axons regenerate into and through the graft; however, they stop when they reach the glial scar at the distal graft-host interface that is rich with inhibitory chondroitin sulfate proteoglycans. We previously showed that treatment of a spinal cord injury site with chondroitinase (ChABC) allows axons within the graft to traverse the scar and reinnervate spinal cord, where they form functional synapses. While this improvement in outgrowth was significant, it still represented only a small percentage (<20%) of axons compared to the total number of axons that regenerated into the PNG. Here we tested whether providing exogenous brain-derived neurotrophic factor (BDNF) via lentivirus in tissue distal to the PNG would augment regeneration beyond a ChABC-treated glial interface. We found that ChABC treatment alone promoted axonal regeneration but combining ChABC with BDNF-lentivirus did not increase the number of axons that regenerated back into spinal cord. Combining BDNF with ChABC did increase the number of spinal cord neurons that were trans-synaptically activated during electrical stimulation of the graft, as indicated by c-Fos expression, suggesting that BDNF overexpression improved the functional significance of axons that did reinnervate distal spinal cord tissue.

Improved Wound Stability of Top-Hat Profiled Femtosecond Laser-Assisted Penetrating Keratoplasty In Vitro

The femtosecond laser is a new option for cutting corneal tissue at high precision. The "top-hat" profile has an overlapping graft-host interface at the edge because of a larger inner trephination diameter. This may enhance graft fixation, thus improving the outcome and accelerating rehabilitation after penetrating keratoplasty. Femtosecond laser top-hat keratoplasties with overlaps of 0 and 3 mm between inner and outer trephination diameters were performed in vitro. After trephination, the excised corneal buttons were readapted by different suturing profiles. Pressure in the artificial anterior chamber was then raised until we observed wound leakage and ultimately wound prolapse. Better wound stability was found in conjunction with all profiled trephinations. When using 4 interrupted sutures, wound leakage occurred at a median of 13.0 cm H2O (mean, 12.3 cm H2O) and "zero overlap," at 19.0 cm H2O (mean, 20.8 cm H2O) and 1-mm overlap, at 32.0 cm H2O (mean, 32.8 cm H2O) and 2-mm overlap, and at 48.5 cm H2O (mean, 49.4 cm H2O) and 3-mm overlap. Comparing zero overlap with the mean values of 1- to 3-mm overlaps, wound leakage happened at 13.0 (mean, 12.3) versus 32.0 (mean, 34.3) cm H2O with 4 interrupted sutures, at 57.5 (mean, 58.3) versus 61.0 (mean, 70.8) cm H2O with 8 interrupted sutures, at 31.5 (mean, 32.0) versus >97.0 (mean, 75.5) cm H2O with 1 running and 4 interrupted sutures, and at 34.0 (mean, 32.3) versus 80.0 (mean, 69.9) cm H2O with 1 running suture. The analysis of variance revealed a statistically significant increase in wound stability for all overlaps independently from the size of the overlap. Femtosecond laser-assisted profiles with even small overlaps for penetrating keratoplasty may make fewer sutures and earlier suture removal possible because of better wound stability, contributing to earlier visual recovery and helping to prevent wound rupture after trauma. However, further study is required to identify the optimum profile including the various technical parameters.

Candida interface keratitis following deep anterior lamellar keratoplasty

The purpose is to report a case of Candida interface infection after deep anterior lamellar keratoplasty (DALK). A 23-year-old female patient underwent DALK surgery in the left eye for keratoconus. Four weeks after the surgery, she presented with asymptomatic white-cream colored deposits at the graft-host interface. Epithelial ingrowth was our first possible diagnosis because there were no symptoms or signs of inflammation. However, progression of the lesion under steroid treatment and the appearance of inflammation signs after tapering the steroid treatment raised suspicion of fungal keratitis. Anterior segment optical coherence tomography (OCT), ultrasound biomicroscopy, confocal microscopy and microbiologic examinations of the cornea were performed to evaluate the lesion. Anterior segment OCT and ultrasound biomicroscopy confirmed the lesion to be at the interface. The confocal scan disclosed hyper-reflective deposits and surrounding inflammatory cells but there were no hyphae-like structures. While taking a specimen from the lesion, the Descemet's membrane ruptured so a penetrating keratoplasty was performed. The microbiologic examination revealed Candida infection. Candida interface keratitis is a rare infection seen after DALK. The asymptomatic clinical picture and the similarity to epithelial ingrowth may postpone the diagnosis and consequently the treatment. Therefore, in cases of interface deposits seen after lamellar surgery, one should consider Candida interface keratitis.

Retained Interface Gentian Violet Ink in Descemet Stripping Endothelial Keratoplasty

To present a case of a patient who underwent right Descemet stripping endothelial keratoplasty (DSEK) in whom the gentian violet ink used to mark and orient the donor corneal graft had persisted at the host-graft interface. A 57-year-old woman with progressive corneal edema from Fuchs endothelial dystrophy in her right eye underwent a combined phacoemulsification/posterior chamber intraocular lens insertion and DSEK. The host endothelium was stripped in a central 9.0-mm diameter. The endothelial graft was harvested to a depth of 400 μm. The anterior stromal flap was lifted, and the stromal side of the endothelial graft was peripherally marked with a dotted "7" using a Codman gentian violet marker pen and a dot was placed at the center to aid centration. The graft was inserted and centrally positioned. The superior clear corneal wound was closed with 3 × 10/0 nylon interrupted sutures. The patient was instructed to lie face up for the first 48 hours. At 1 week, the graft was well positioned and the central gentian violet mark was not present but the peripheral markings were noted. At 14 months, the cornea was clear with an unaided visual acuity of 20/25 and best-corrected visual acuity of 20/20 but the superior peripheral markings persisted at the graft-host interface. Specular microscopy demonstrated a cell density of 1900 cells per square millimeter. Gentian violet ink marking used in DSEK operations may persist at the graft-host interface. Small, peripheral markings will minimize the risk of interference with central vision, ink-induced inflammation, and endothelial damage.