In vivo confocal microscopy and optical coherence tomography as innovative tools for the diagnosis of limbal stem cell deficiency.

The limbus is the anatomical and functional barrier between the corneal and conjunctival epithelia. It is characterized by the presence of the limbal stem cell niche, which allows corneal homeostasis to be maintained. Limbal stem cell deficiency is characterized by a dual process: insufficient regeneration of corneal epithelium, which cannot therefore assure its function of physiological support, associated with corneal invasion by conjunctival proliferation. Diagnosis is currently made via routine clinical examination, corneal impression cytology and in vivo confocal microscopy (IVCM). Slit lamp examination shows abnormal limbal anatomy, thin and irregular epithelium with late fluorescein staining, and superficial vascularization. With its high resolution, IVCM allows identification of limbal and corneal epithelial changes at a cellular level in en face views parallel to the corneal surface, but with a restricted viewing field of the corneal surface. It shows a poor transition between the corneal and conjunctival epithelia, associated with a loss of the normal corneal epithelial stratification, low basal cell and sub-basal nerve plexus densities, and subepithelial fibrosis. Spectral domain optical coherence tomography of the central cornea and limbus, with scans in variable orientations, allows a quick, global and non-invasive analysis of normal eyes and those with limbal stem cell deficiency. It shows a thin limbal epithelium, lacking normal thickening, featuring absence of stromal undulations and limbal crypts in cross-sections and sections parallel to the limbus, lack of visible limbal crypts in en face sections, loss of clear transition between the hyporeflective corneal epithelium and the hyperreflective conjunctival epithelium, and hyperreflective subepithelial fibrosis. The limbus is the anatomical and functional barrier between the corneal and conjunctival epithelia. It is characterized by the presence of the limbal stem cell niche, which allows corneal homeostasis to be maintained. Limbal stem cell deficiency (LSCD) is characterized by a dual process: insufficient regeneration of corneal epithelium, which cannot therefore assure its function of physiological support, associated with corneal invasion by conjunctival proliferation.

[In vivo confocal microscopy and optical coherence tomography as innovative tools for the diagnosis of limbal stem cell deficiency (French translation of the article)]. / Progrès dans les méthodes diagnostiques du déficit en cellules souches limbiques. Apport de la microscopie confocale et de la tomographie en cohérence optique.

The limbus is the anatomical and functional barrier between corneal and conjunctival epithelia. It is characterized by presence of the limbal stem cell niche which allows corneal homeostasis to be maintained. Limbal stem cell deficiency is characterized by a dual process: insufficient regeneration of corneal epithelium, which cannot therefore assure its function of physiological support, associated with corneal invasion by conjunctival proliferation. Diagnosis is currently made via routine clinical examination, corneal impression cytology and in vivo confocal microscopy (IVCM). Slit lamp examination shows abnormal limbal anatomy, thin and irregular epithelium with late fluorescein staining, and superficial vascularization. With its high resolution, IVCM allows identification of limbal and corneal epithelial changes at a cellular level in en face views, parallel to the corneal surface, but with a restricted viewing field of the corneal surface. It shows a poor transition between the corneal and conjunctival epithelia, associated with a loss of the normal corneal epithelial stratification, low basal cell and sub-basal nerve plexus densities, even with sub-epithelial fibrosis. Optical coherence tomography in central cornea and at the limbus, with scans in different orientations, allows a quick, global and non-invasive analysis of normal eyes and those with limbal stem cell deficiency. It shows a thin limbal epithelium, lacking normal thickening, featuring absence of stromal undulations and limbal crypts in cross-sections and sections parallel to the limbus, lack of visible limbal crypts in en face sections, loss of clear transition between the hyporeflective corneal epithelium and the hyperreflective conjunctival epithelium, and hyperreflective sub-epithelial fibrosis.

Simultaneous penetrating keratoplasty and amniotic membrane transplantation in eyes with a history of limbal stem cell deficiency.

PURPOSE: To describe the outcomes of simultaneous penetrating keratoplasty (PK) and amniotic membrane transplantation (AMT) performed both as a ring-shaped graft and as a temporary patch in eyes with a history of limbal stem cell deficiency (LSCD). METHODS: Prospective observational case series including 48 simultaneous PK/AMT procedures (48 patients) in eyes with a history of partial or total LSCD. Patients with total LSCD were first treated with limbal stem cell transplantation. The preoperative indication was graft failure in 58.3% of cases. Most recipients (89.6%) were at high-risk for rejection. RESULTS: The mean graft reepithelialization time was 29.2±30.8 days. Graft reepithelialization was achieved in 30 days in 70.8% of cases. No AMT-related adverse events were observed. The mean time from keratoplasty-to-last visit was 84.5±54.5 months. The 3-year graft survival rate was 62.5%. Recurrence of corneal epithelial defects after graft reepithelialization (47.9%) was associated with lower graft survival (P=0.004). In eyes with successful grafts at the last visit, the mean LogMAR visual acuity was 1.90 (20/1575)±5 lines before keratoplasty and 0.89 (20/155)±10 lines at 5 years. A ring of amniotic membrane was visible between the graft stroma and the corneal epithelium on slit-lamp examination and optical coherence tomography in all successful cases. CONCLUSIONS: In this series of eyes with a history of LSCD and at high-risk of rejection, simultaneous PK and AMT were associated with satisfactory graft survival and no additional adverse events.

[Corneal collagen cross-linking to treat infectious keratitis]. / Le cross-linking du collagène dans le traitement des infections cornéennes.

Infectious keratitis are a frequent cause of ocular morbidity. Today, new treatments are necessary to combat the emergence of antibiotic resistant germs. Corneal collagen cross-linking has been suggested to treat corneal infectious (PACK-CXL). Its action would be both antimicrobial and protective for the cornea, increasing its biochemical resistence to proteolytic enzymes. In vivo, PACK-CXL might demonstrate good efficacy against bacterial keratitis, contrary to herpetic keratitis for which it is contraindicated. For fungal or amoebic keratitis, results are uncertain regarding its safety and efficacy. The purpose of this paper is to clarify the use of corneal collagen cross-linking to treat infectious keratitis.

Three-dimensional structure of the mammalian limbal stem cell niche.

Although the existence of the limbal stem cell (LSC) niche is accepted, precise knowledge of its three-dimensional (3D) architecture remains incomplete. The LSC niche was explored on freshly excised and organ-cultured corneoscleral rims from human donors (n = 47), pigs (n = 15) and mice (n = 27) with full-field optical coherence microscopy (FFOCM). Limbal crypt features were detected in 90% of organ-cultured human corneoscleral rims, extending between the palisades of Vogt as radially oriented rectangular (74% of eyes) and/or rounded (23% of eyes) forms, often branching off to, or becoming interconnected by, sub-scleral radially or circumferentially oriented crypts (in 56% of eyes). Mean crypt volume represented 16% of sampled limbal volume on the vertical axis and 8% on the horizontal axis. In pigs, palisades were finer and crypts wider with relatively uniform distribution around the eye, and radial orientation, connecting to numerous narrow criss-crossing invaginations beneath the scleral surface. In mice, only a circumferential limbal trough was detected. Mean crypt volume represented 13% of sampled limbal volume in humans and 9% in pigs. FFOCM combined with fluorescence, and confocal fluorescence microscopy, showed presence of p63-α+ cells and cytokeratin-3+ cells in the limbal crypts. To assess colony forming efficiency (CFE), limbal epithelial cells were cultured at low density with mitomycin-arrested 3T3 feeders. CFE increased with limbal crypt volume and was not significantly decreased in organ-cultured cornea, despite degradation of the epithelial roof, suggesting that stem cells remain protected at the base of crypts during organ culture. CFE in human samples was significantly greater than in pig, and CFE in mouse was zero. Crypt architecture in the three species appears associated with eye exposure to light. LSC density increased with percentage limbal volume occupied by crypts.

[Influence of surgical technique on graft and endothelial survival in endothelial keratoplasty]. / Influence des techniques chirurgicales de greffe endothéliale sur la survie du greffon et de son endothélium.

PURPOSE: To report survival of the graft and its endothelium after Descemet Stripping Endothelial Keratoplasty (DSEK) in a series of consecutive cases with no exclusion of cases corresponding to the learning curve and to analyze the influence of surgical techniques on survival. PATIENTS AND METHODS: This prospective observational study includes 170 consecutive DSEK's performed between 2006 and 2013. The main outcome criteria were graft survival and survival of the donor corneal endothelium as assessed by specular microscopy. The following parameters were analyzed: preoperative diagnosis, lens status, surgical techniques, and graft thickness. RESULTS: Graft survival was 91.7% at 1 year and 71.5% at 3 years. Graft survival was significantly associated with surgical technique (P=0.04). The best graft survival was achieved with scleral incision combined with graft insertion with the Endosaver® device (dedicated DSEK injector). Graft survival decreased with graft thickness (P<0.001). One-year endothelial cell density was significantly associated with surgical technique (P=0.003). Early 1-year endothelial cell loss was 42.0% for the scleral incision/Endosaver® group, 48.7% for the corneal incision/Endosaver® group, 49.4% for the corneal incision/Busin guide group, 66.0% for the corneal incision/IOL injector group, and 66.7% for the scleral incision/forceps group (P=0.002). CONCLUSION: The success rate of DSEK is close to that of penetrating keratoplasty. The use of a DSEK-dedicated injector results in higher survival of the graft and its endothelium. The use of ultrathin grafts also appears to represent significant progress.