Björn Bachmann

Cutting Edge: Lymphatic Vessels, Not Blood Vessels, Primarily Mediate Immune Rejections After Transplantation

The purpose of this study was to determine the relative importance of blood vessels (hemangiogenesis) versus lymphatic vessels (lymphangiogenesis) in mediating immunological responses after transplantation. Using the murine model of corneal transplantation, graft survival was compared in differentially prevascularized and avascular recipient beds. Donor corneas (C57BL/6) were transplanted into uninflamed or inflamed avascular, prehemvascularized only or prehemvascularized and prelymphvascularized recipient murine eyes (BALB/C). Selective inhibition of lymphangiogenesis was achieved using antivascular endothelial growth factor receptor 3 Abs and anti-integrin α5 small molecules. Grafts placed into only prehemvascularized recipient beds had a similarly good graft survival compared with grafts placed into completely avascular, normal recipients, whereas the pre-existence of lymphatic vessels significantly deteriorated corneal graft survival (p < 0.05). Lymphatic vessels seem to contribute significantly to graft rejection after (corneal) transplantation. That may allow for selective, temporary, perioperative antilymphangiogenic treatment to promote graft survival without affecting blood vessels, even after solid organ transplantation.

A stepwise approach to donor preparation and insertion increases safety and outcome of Descemet membrane endothelial keratoplasty.

Purpose: Lamellar techniques for selective replacement of diseased corneal structures have recently been improved. Descemet membrane endothelial keratoplasty (DMEK) allows the sole replacement of the endothelium-Descemet membrane layer (EDM). However, widespread use of DMEK is currently limited because of problems with donor preparation namely the tearing of the Descemet membrane and the difficulty to unfold the EDM graft in the anterior chamber (AC). Methods: A standardized DMEK procedure that allows safe preparation of EDM, atraumatic introduction of EDM into the AC, reliable orientation of EDM during surgery, and stepwise unfolding within the AC is described in 80 patients. Visual acuity and corneal endothelial cell density were assessed. Results: A stepwise approach using a novel bimanual underwater technique to harvest EDM from donor corneal buttons allows reproducible generation of grafts without tearing the Descemet membrane. Injection of the EDM roll into the AC is achieved by use of a standard injector cartridge, whereas the depth of AC is maintained by an irrigation handpiece. Marks at the margin of EDM allow orientation. Finally, unfolding EDM in the AC is achieved by sequential use of water jets and air bubbles. In the early phase of the learning curve, 4 patients were regrafted because of graft failure. Endothelial cell density decreased from 2600 ± 252 to 1526 ± 341 cells per square millimeter 1 month after DMEK. Conclusions: A novel technique for graft preparation and EDM injection results in improved safety with a high rate of successful DMEKs.

Corneal Limbal Microenvironment Can Induce Transdifferentiation of Hair Follicle Stem Cells into Corneal Epithelial‐like Cells

The aim of this study was to investigate the transdifferentiation potential of murine vibrissa hair follicle (HF) stem cells into corneal epithelial‐like cells through modulation by corneal‐ or limbus‐specific microenvironmental factors. Adult epithelial stem cells were isolated from the HF bulge region by mechanical dissection or fluorescence‐activated cell sorting using antibodies to α6 integrin, enriched by clonal expansion, and subcultivated on various extracellular matrices (type IV collagen, laminin‐1, laminin‐5, fibronectin) and in different conditioned media derived from central and peripheral corneal fibroblasts, limbal stromal fibroblasts, and 3T3 fibroblasts. Cellular phenotype and differentiation were evaluated by light and electron microscopy, real‐time reverse transcription‐polymerase chain reaction, immunocytochemistry, and Western blotting, using antibodies against putative stem cell markers (K15, α6 integrin) and differentiation markers characteristic for corneal epithelium (K12, Pax6) or epidermis (K10). Using laminin‐5, a major component of the corneo‐limbal basement membrane zone, and conditioned medium from limbal stromal fibroblasts, clonally enriched HF stem and progenitor cells adhered rapidly and formed regularly arranged stratified cell sheets. Conditioned medium derived from limbal fibroblasts markedly upregulated expression of cornea‐specific K12 and Pax6 on the mRNA and protein level, whereas expression of the epidermal keratinocyte marker K10 was strongly downregulated. These findings suggest that adult HF epithelial stem cells are capable of differentiating into corneal epithelial‐like cells in vitro when exposed to a limbus‐specific microenvironment. Therefore, the HF may be an easily accessible alternative therapeutic source of autologous adult stem cells for replacement of the corneal epithelium and restoration of visual function in patients with ocular surface disorders. STEM CELLS 2009;27:642–652

Corneal Neovascularization as a Risk Factor for Graft Failure and Rejection after Keratoplasty: An Evidence-Based Meta-analysis

TOPIC Preoperative corneal neovascularization (CNV) is thought to be associated with an increased rate of corneal graft failure and potentially also graft rejection. CLINICAL RELEVANCE New therapeutic options that offer differential influence on the ingrowths or regression of either corneal blood or lymphatic vessels force us to re-evaluate the known data about the role of CNV in keratoplasty. METHODS Electronic databases and corneal registries were searched (up through September 2008). Results were reported both descriptively for each study and using random effects meta-analysis. Potential moderating factors for the association between vascularization and graft failure and rejection were examined using metaregression analysis. RESULTS Nineteen studies reporting on a total of 24,944 grafts undergoing keratoplasty were included. An increase in the risk of graft failure and rejection in the presence of pathologic CNV was seen in studies with a pooled risk ratio of 1.32 (95% confidence interval [CI], 1.15-1.49) for graft failure and 2.07 (95% CI, 0.98-3.15) for graft rejection. There was evidence of incremental increase of risk for graft failure and rejection as more corneal quadrants were affected by neovascularization. The 2 factors predictive of increased risk of neovascularization and graft failure were increased recipient age (P = 0.003) and male gender (P = 0.046). CONCLUSIONS Graft failure and rejection risk increase with an increasing number of corneal quadrants affected by neovascularization before keratoplasty. These data support the study of novel topical antiangiogenic therapies at the cornea to precondition such a cornea for future corneal grafting. FINANCIAL DISCLOSURE(S) Proprietary or commercial disclosure may be found after the references.

Safety profile of topical VEGF neutralization at the cornea.

PURPOSE Bevacizumab eyedrops inhibit corneal neovascularization. The purpose of this study was to analyze the safety profile of VEGF-A neutralization at the ocular surface. METHODS Bevacizumab eyedrops (5 mg/mL) and an antimurine VEGF-A antibody (250 microg/mL) were applied to normal murine corneas five times a day for 7 and 14 days. Subsequently, corneas were analyzed for morphologic changes by light and electron microscopy. In a mouse model of corneal epithelial abrasion, the effects of topically applied anti-VEGF antibodies on epithelial wound healing were analyzed: the treatment group received bevacizumab (5 mg/mL) or the antimurine VEGF-A antibody (250 microg/mL) as eyedrops, and the control group received an equal volume of saline solution. After 12, 18, and 24 hours, corneas were photographed in vivo with and without fluorescein staining for morphometry. Afterwards the mice were killed, and eyes were removed for histology, immunohistochemistry with Ki67/DAPI, and electron microscopy. The effect of midterm anti-VEGF therapy on corneal nerve density was assessed by staining corneas treated with an FITC-conjugated anti-neurofilament antibody and morphometric analysis. RESULTS Murine corneas treated with two different types of anti-VEGF antibody eyedrops did not show obvious corneal morphologic changes at the light and electron microscopic levels. Furthermore, anti-VEGF antibody eyedrops had no significant impact on the wound healing process after corneal epithelial injury or on normal murine corneal nerve fiber density. CONCLUSIONS Topical neutralization of VEGF-A at the corneal surface does not have significant side effects on normal corneal epithelial wound healing, normal corneal integrity, or normal nerve fiber density. Therefore, anti-VEGF eyedrops seem to be a relatively safe option to treat corneal neovascularization.

Aganirsen Antisense Oligonucleotide Eye Drops Inhibit Keratitis-Induced Corneal Neovascularization and Reduce Need for Transplantation: The I-CAN Study

OBJECTIVE Eye drops of aganirsen, an antisense oligonucleotide preventing insulin receptor substrate-1 expression, inhibited corneal neovascularization in a previous dose-finding phase II study. We aimed to confirm these results in a phase III study and investigated a potential clinical benefit on visual acuity (VA), quality of life (QoL), and need for transplantation. DESIGN Multicenter, double-masked, randomized, placebo-controlled phase III study. PARTICIPANTS Analysis of 69 patients with keratitis-related progressive corneal neovascularization randomized to aganirsen (34 patients) or placebo (35 patients). Patients applied aganirsen eye drops (86 μg/day/eye) or placebo twice daily for 90 days and were followed up to day 180. MAIN OUTCOME MEASURES The primary end point was VA. Secondary end points included area of pathologic corneal neovascularization, need for transplantation, risk of graft rejection, and QoL. RESULTS Although no significant differences in VA scores between groups were observed, aganirsen significantly reduced the relative corneal neovascularization area after 90 days by 26.20% (P = 0.014). This improvement persisted after 180 days (26.67%, P = 0.012). Aganirsen tended to lower the transplantation need in the intent-to-treat (ITT) population at day 180 (P = 0.087). In patients with viral keratitis and central neovascularization, a significant reduction in transplantation need was achieved (P = 0.048). No significant differences between groups were observed in the risk of graft rejection. However, aganirsen tended to decrease this risk in patients with traumatic/viral keratitis (P = 0.162) at day 90. The QoL analyses revealed a significant improvement with aganirsen in composite and near activity subscores (P = 0.039 and 0.026, respectively) at day 90 in the per protocol population. Ocular and treatment-related treatment-emergent adverse events (TEAEs) were reported in a lower percentage with aganirsen compared with placebo. Only 3 serious TEAEs (2 with aganirsen and 1 with placebo) were considered treatment-related. CONCLUSIONS This first phase III study on a topical inhibitor of corneal angiogenesis showed that aganirsen eye drops significantly inhibited corneal neovascularization in patients with keratitis. The need for transplantation was significantly reduced in patients with viral keratitis and central neovascularization. Topical application of aganirsen was safe and well tolerated.

Microbubble incision as a new rescue technique for big-bubble deep anterior lamellar keratoplasty with failed bubble formation.

Purpose: To describe a new surgical technique allowing dissection down to Descemet membrane in big-bubble deep anterior lamellar keratoplasty (DALK) with failed big-bubble formation (the “microbubble incision technique”). Methods: This is an interventional case series of 10 consecutive patients with keratoconus undergoing intended big-bubble DALK with failure to establish a normal big bubble. In all patients, repeated air injections into the stroma were performed, leaving a whitish colored stroma. Lamellar dissection as far down as possible was then performed within this white tissue. As soon as the anterior chamber was visible, a large remaining intrastromal air bubble was incised with a sharp 15-degree knife introduced perpendicular to the tissue to open up this predescemetic bubble. If deeper air bubbles were still visible, this approach was repeated. Using a blunt spatula, this new layer was then prepared and viscodissection performed. Results: Using this novel approach, in 9 of the 10 patients, it was possible to dissect down to Descemet membrane. Macroperforation made conversion to penetrating keratoplasty necessary in 1 patient. Microperforations not necessitating conversion occurred in 2 patients. All 9 patients with “rescued” DALK had an uneventful postoperative course and had a mean visual acuity of 20/63 ± 20/125 (range, 20/500–20/50) and a mean endothelial cell count of 1672 ± 163 cells per square millimeter (range, 1493–1867 cells/mm2) at 3 months. Conclusions: Microbubble incision is a new rescue technique for big-bubble DALK patients without bubble formation allowing for a safer dissection down to Descemet membrane.

Patient Satisfaction after Posterior Lamellar Keratoplasty (DSAEK)

BACKGROUND The advantages of posterior lamellar keratoplasty (DSAEK) compared to conventional full thickness keratoplasty are faster visual rehabilitation, little change in astigmatism and the absence of suture-associated complications. The current study evaluated the satisfaction of the first 15 patients who underwent DSAEK at our clinic. MATERIALS AND METHODS Evaluation of patient satisfaction was conducted via a telephone survey and by means of a written patient questionnaire. The patients were asked to answer questions on a scale from 1 (very bad) to 10 (very good) about their overall satisfaction with this procedure, their satisfaction with the achieved postoperative visual acuity, their satisfaction with the progress of the healing process and the extent of the perceived burden. The patients were also asked whether they would opt again for the operation. Pre- and postoperative visual acuity, IOP, topographic astigmatism and corneal pachymetry were analysed. RESULTS 15 eyes of 15 patients were analysed (12 women and 3 men). Average age at the time of operation was 75 +/- 7 years. Visual acuity increased and corneal thickness decreased primarily within the first three months. During that time there was an increase in overall patient satisfaction which finally reached 6.9 +/- 1.8 on the scale. Postoperative visual acuity was awarded with 7.2 +/- 2.0, progress of the healing process with 7.4 +/- 2.3. The procedure was not perceived as a big burden (8.0 +/- 1.9) and most patients would have decided again for the operation (8.2 +/- 2.0). There was no pre- and postoperative difference in IOP and corneal astigmatism. CONCLUSIONS Like in our group patients with diseases of the corneal endothelium are of higher age and therefore benefit from a fast restitution of visual acuity and a low burden of operation. DSAEK represents a secure and practical procedure to rehabilitate elderly patients with diseases of the corneal endothelium.

Ocular manifestations in the mucopolysaccharidoses – a review

Ocular manifestations are very common in all types of mucopolysaccharidoses (MPS) and often lead to visual impairment. They arise as a result of the accumulation of glycosaminoglycans deposits in ocular tissues or secondary to increased intracranial pressure. Typical ocular features in MPS include corneal clouding, retinopathy, glaucoma, optic disc swelling, optic atrophy, and ocular motility and refractive error problems. This paper reviews the ocular features in patients with MPS, discusses the diagnosis of these ocular features and the diagnostic problems that may arise in patients (children) with MPS, and highlights the central role ophthalmologists may play in the diagnosis and follow‐up of these patients.

Novel collagen membranes for the reconstruction of the corneal surface.

No standardized biomaterial exists for the surgical treatment of persistent corneal erosions and ulcerations. We analyzed the suitability and biocompatibility of defined noncross-linked and UV/riboflavin cross-linked equine type I collagen membranes for the reconstruction of the corneal surface. Isolated human oral mucosa epithelial cells, a cell type in clinical use for the treatment of ocular surface diseases, were subcultivated on both types of membranes and examined concerning cell adhesion, proliferation, and differentiation. Biocompatibility was evaluated following superficial and intrastromal corneal transplantation in New Zealand white rabbits. In cell cultures all collagen membranes supported adhesion of oral mucosa epithelial cells leading to the formation of multilayered epithelial cell sheets. After intrastromal corneal implantation clinical signs of degradation were seen in all variants of collagen membranes, which was fastest in noncross-linked variants. The histological and ultrastructural level invasion of keratocytes and production of new collagen fibers inside the collagen membranes could be detected in noncross-linked variants. After superficial corneal implantation covering of the membranes by corneal epithelium over time was visible. Ultrastructural analysis showed a slower rate of degradation and less invading keratocytes in cross-linked variants compared with noncross-linked collagen membranes. Cross-linked and noncross-linked variants of the collagen membrane proofed to be suitable to serve as a carrier for epithelial stem cells in vitro and showed a high biocompatibility in vivo. These results indicate that the tested collagen membranes might be suitable for the reconstruction of the corneal surface in patients with nonhealing ulcerations. Whether membranes with faster or slower degradation properties are preferable for the treatment of persistent corneal ulcerations might depend on the underlying corneal pathology and the degree of concomitant inflammation.