Development of ex vivo organ culture models to mimic human corneal scarring.

PURPOSE: To develop ex vivo organ culture models of human corneal scarring suitable for pharmacological testing and the study of the molecular mechanisms leading to corneal haze after laser surgery or wounding. METHODS: Corneas from human donors were cultured ex vivo for 30 days, either at the air-liquid interface (AL) or immersed (IM) in the culture medium. Histological features and immunofluorescence for fibronectin, tenascin C, thrombospondin-1, and α-smooth muscle actin were graded from 0 to 3 for control corneas and for corneas wounded with an excimer laser. The effects of adding 10 ng/ml transforming growth factor-ß1 (TGF-ß1) to the culture medium and of prior complete removal of the epithelium and limbus, thus preventing reepithelialization, were also analyzed on wounded corneas. Collagen III expression was detected with real-time PCR. RESULTS: Wounding alone was sufficient to induce keratocyte activation and stromal disorganization, but it was only in the presence of added TGF-ß1 that intense staining for fibronectin and tenascin C was found in the AL and IM models (as well as thrombospondin-1 in the AL model) and that α-smooth muscle actin became detectable. The scar-like appearance of the corneas was exacerbated when TGF-ß1 was added and reepithelialization was prevented, resulting in the majority of corneas becoming opaque and marked upregulation of collagen III. CONCLUSIONS: THE MAIN FEATURES OF CORNEAL SCARRING WERE REPRODUCED IN THESE TWO COMPLEMENTARY MODELS: the AL model preserved differentiation of the epithelium and permits the topical application of active molecules, while the IM model ensures better perfusion by soluble compounds.

Acute Retinal Necrosis: Virological Features Using Quantitative Polymerase Chain Reaction, Therapeutic Management, and Clinical Outcomes.

PURPOSE: To evaluate outcomes of patients treated with intensive intravitreal therapy and to describe the evolution of quantitative real-time polymerase chain reaction (qPCR) in patients treated for acute retinal necrosis (ARN) syndrome. DESIGN: Retrospective observational case series. METHODS: This study included 25 eyes of 24 patients with ARN who were treated and followed up in 2 departments of ophthalmology in Lyon, France. Assessed outcomes included qPCR viral load profile during treatment, number of antiviral intravitreal injections (IVT), retinal detachment rate, and best-corrected visual acuity. RESULTS: Final visual acuity was 20/200 or less in 20% of cases; the rate of retinal detachment was 16%. Viral load kinetics changed in 3 phases: a first plateau period that was not consistent, a logarithmic decrease phase, and a negativation phase. Mean decay of the logarithm of the viral load was estimated at 0.076 per day; mean time of negativation was 56.1 days. Median IVT number was 9 (range, 0-28). Ten patients were treated with injections until the viral load was undetectable. Resistance to acyclovir was observed in a patient with a prolonged initial plateau of the viral load. CONCLUSIONS: Numerous and prolonged IVTs, used as adjunctive therapy, could improve the prognosis of treated patients by decreasing the risk of retinal detachment and improving visual acuity. QPCR enables monitoring of the response to treatment and can provide evidence for resistance to antiviral treatment by enabling the detection of cases with a prolonged initial plateau of viral load.

Incidence of Cystoid Macular Edema After Descemet Membrane Endothelial Keratoplasty.

PURPOSE: The incidence of and risk factors for cystoid macular edema (CME) after Descemet membrane endothelial keratoplasty (DMEK) remain uncertain. This study examines the incidence of and risk factors for CME after DMEK. METHODS: This retrospective, single-center study included patients with no history of CME who had undergone DMEK. Patients were examined weekly for 1 month after surgery and at 3 and 6 months after surgery. Follow-up examinations included visual acuity (VA) assessment, pachymetry, anterior segment optical coherence tomography, biomicroscopy, intraocular pressure measurement, and fundoscopy. Eyes suspected of having CME (reduced VA and/or abnormal fundoscopic findings) underwent macular optical coherence tomography. Potential risk factors for CME examined included age, axial length, anterior chamber rebubbling, not using a topical nonsteroidal antiinflammatory after surgery, and concurrent DMEK and cataract surgery (triple-DMEK). RESULTS: Eighty eyes (74 subjects) were included. Eleven eyes (13.8%) developed CME within 6 months after undergoing DMEK. Univariate analyses did not identify any significant CME risk factors. Interestingly, the triple-DMEK procedure did not put subjects at risk for developing CME (P = 0.184). Visual prognosis after medical treatment for CME was excellent, and subjects with and without CME had comparable VA at 6 months [CME: logarithm of the minimum angle of resolution (logMAR) VA = 0.3 (first-third quartile: 0.1-1.0), 20/40; no CME: logMAR VA = 0.3 (0.1-0.5), 20/40; P = 0.391]. CONCLUSIONS: Although CME frequently occurred after DMEK, no CME risk factors were identified. In addition, CME did not significantly affect long-term visual outcomes when it was appropriately treated.

Use of magnetically oriented orthogonal collagen scaffolds for hemi-corneal reconstruction and regeneration.

We recently showed that the highly organized architecture of the corneal stroma could be reproduced using scaffolds consisting of orthogonally aligned multilayers of collagen fibrils prepared using a high magnetic field. Here we show that such scaffolds permit the reconstruction in vitro of human hemi-corneas (stroma + epithelium), using primary human keratocytes and limbal stem cell derived human keratinocytes. On the surface of these hemi-corneas, a well-differentiated epithelium was formed, as determined both histologically and ultrastructurally and by the expression of characteristic markers. Within the stroma, the keratocytes aligned with the directions of the fibrils in the scaffold and synthesized a new extracellular matrix with typical collagen markers and small, uniform diameter fibrils. Finally, in vivo experiments using a rabbit model showed that these orthogonally oriented multi-layer scaffolds could be used to repair the anterior region of the stroma, leading to re-epithelialization and recovery of both transparency and ultrastructural organization.

Bacterial adhesion to conventional hydrogel and new silicone-hydrogel contact lens materials.

BACKGROUND: As bacterial adhesion to contact lenses may contribute to the pathogenesis of keratitis, the aim of our study was to investigate in vitro adhesion of clinically relevant bacteria to conventional hydrogel (standard HEMA) and silicone-hydrogel contact lenses using a bioluminescent ATP assay. METHODS: Four types of unworn contact lenses (Etafilcon A, Galyfilcon A, Balafilcon A, Lotrafilcon B) were incubated with Staphylococcus epidermidis (two different strains) and Pseudomonas aeruginosa suspended in phosphate buffered saline (PBS). Lenses were placed with the posterior surface facing up and were incubated in the bacterial suspension for 4 hours at 37 degrees C. Bacterial binding was then measured and studied by bioluminescent ATP assay. Six replicate experiments were performed for each lens and strain. RESULTS: Adhesion of all species of bacteria to standard HEMA contact lenses (Etafilcon A) was found to be significantly lower than that of three types of silicone-hydrogel contact lenses, whereas Lotrafilcon B material showed the highest level of bacterial binding. Differences between species in the overall level of adhesion to the different types of contact lenses were observed. Adhesion of P. aeruginosa was typically at least 20 times greater than that observed with both S. epidermidis strains. CONCLUSIONS: Conventional hydrogel contact lenses exhibit significantly lower bacterial adhesion in vitro than silicone-hydrogel ones. This could be due to the greater hydrophobicity but also to the higher oxygen transmissibility of silicone-hydrogel lenses.