[Corneal collagen crosslinking in keratoconus]. / Le crosslinking du collagène dans le kératocône.

Corneal collagen crosslinking (CXL) is, at present, the only treatment that can slow or even stop the progression of keratoconus. It uses riboflavin and ultraviolet A (UVA) to create covalent bonds ("crosslinks") between collagen fibrils thus increasing corneal rigidity. Although to date there has been no direct evidence of intrastromal corneal crosslinking, several studies have reported the safety and efficacy of the conventional CXL protocol. This protocol is indicated for progressive keratoconus with a minimal corneal thickness (without the epithelium) of at least 400 µm. It should be performed as early as possible in patients under 18 years with keratoconus or with post-LASIK ectasia. Because of the epithelial debridement, it may rarely induce complications such as infectious keratitis or stromal scars. A variety of new protocols is under investigation and may reduce the rate of these complications. In addition, combination of CXL with other surgical treatments (intracorneal ring segments or photorefractive keratectomy) may improve visual outcomes in patients with keratoconus. Finally, the antimicrobial and anti-edematous properties of CXL have been shown, suggesting new therapeutic indications of this procedure such as infectious keratitis or stromal edema in the future.

[Epithelial basement membrane dystrophy diagnosed following LASIK surgery]. / Dystrophie de Cogan révélée après chirurgie réfractive de type Lasik.

A 48-year-old woman with no significant past history underwent bilateral simultaneous laser in situ keratomileusis for correction of her myopia. On the tenth postoperative day, the patient complained of visual decrease and photophobia. Slit lamp exam showed corneal epithelial irregularities. Confocal microscopy was performed and revealed a characteristic appearance of epithelial basement membrane dystrophy (EBMD). The patient was successfully treated with artificial tears and autologous serum eyedrops. EBMD may be missed before LASIK surgery, even after a careful pre-operative examination. Exacerbation of EBMD after LASIK surgery is rare. It should be considered when unexplained corneal epithelial defects or irregularities occur following LASIK. Confocal microscopy is very useful to confirm the diagnosis.

[Recurrence of an idiopathic vasocentric epiretinal membrane: clinical and surgical particularities]. / Récidive d'une membrane épirétinienne idiopathique dite « vasocentrique ¼: particularités cliniques et chirurgicales.

The vasocentric epiretinal membranes (ERM) are idiopathic ERM centered on retinal blood vessels, described mainly in young patients. We report a case of a 70-year-old patient who presented with a decrease in visual acuity secondary to a vasocentric epiretinal membrane. A successful vitrectomy and ERM removal were performed. Four years after surgery, a contractile ERM centered on the superior temporal blood vessel occurred and was associated with retinal distortions at the posterior pole. The second surgery combined removal of the recurrent ERM, which was adherent to the temporal vessels, and peeling of the internal limiting membrane in the macular area. Although there was visual recovery, the patient is still suffering from metamorphopsia 2 years after surgery. The vasocentric ERM have a poor visual outcome and a high risk of recurrence in comparison with other ERM disorders. This case report describes the main clinical and surgical characteristics of this type of membrane.

Human epithelial cell cultures from superficial limbal explants.

PURPOSE: To study the kinetics of growth and the phenotype of cells cultured from human limbal explants in a cholera toxin-free medium with no feeder cell layer. METHODS: Human organ-cultured corneas were used to prepare limbal explants (full-thickness and superficial limbal explants) and corneal stromal explants. Cell growth kinetics and phenotypes were assessed by cultivating explants in cholera toxin-free Green medium. Epithelial and progenitor cell markers were assessed by immunocytochemistry, flow cytometry, and Reverse Transcription and Polymerase Chain Reaction (RT-PCR). RESULTS: The successful epithelial cell growth rates from full thickness limbal explant and superficial limbal explant tissues were 41 and 86%, respectively (p=0.0001). The mean cell area and the percentage of small cells in superficial and full-thickness explant cultures were, respectively, 317 µm(2) and 429 µm(2), and 8.9% and 1.7% (p<0.001). The percentage of positive cells in superficial and full-thickness limbal explant cultures as assessed by immunocytochemistry were the following: broad spectrum cytokeratins (cytokeratins 4, 5, 6, 8, 10, 13, and 18 [MNF116]), 82%/37% (p=0.01); cytokeratin 3 (CK3), 74%/25% (p=0.009); cytokeratin 19 (CK19), 46%/25% (p=0.19); vimentin, 56%/53% (p=0.48); delta N p63α, 54%/0% (p<0.001); and ABCG2, 5%/0% (p=0.1). Flow cytometry showed a higher percentage of small cells, a higher percentage of MNF116+ cells, and stronger expression of progenitor-associated markers in superficial than in full-thickness explant cultures. For superficial limbal explant cultures, analysis of the expression profiles for various mRNAs at the end of 21 days of culture showed high levels of expression of the mRNAs encoding CK3, vimentin, and CK19. The expression of mRNA of delta N p63α and ABCG2 was weaker. Cultures obtained from full-thickness limbal explants featured no expression of mRNA of CK19, delta N p63α, and ABCG2, whereas mRNAs encoding CK3 and vimentin were detected. Human corneal stromal explants cultured with the same medium featured late cell growth, large mean cell area (2,529 µm(2)), no expression of cytokeratins, delta N p63α, and ABCG2, and high expression of vimentin. CONCLUSIONS: Superficial limbal explants appear to be superior to full-thickness limbal explants for growing human limbal epithelial cells. Preparation of explants using surgical facilities (i.e., operating microscope and microsurgical blades) led to a dramatic increase in the percentage of successful cultures, higher epithelial cell growth, decreased fibroblast contamination, and better preservation of limbal epithelial progenitors.

[Human limbal epithelial cell growth kinetics in vitro]. / Cinétique de croissance in vitro des cellules épithéliales cultivées à partir d'explants limbiques humains.

PURPOSE: To assess limbal epithelial cell growth kinetics in vitro using tissue retrieved from organ-cultured donor corneas. PATIENTS AND METHODS: Twenty-one limbal explants were retrieved from corneoscleral rims of donor corneal grafts preserved for 18-24 days in organ culture. The explants were cultured at 37°C for 10, 13, or 18 days. The epithelial cell sheet area was measured during culture by means of morphometry. At the end of culture, the dissociated cells were counted and analyzed by immunocytochemistry. RESULTS: The curve of the epithelial cell sheet area (S) in relation to culture time (t) was best fitted by a polynomial model (S=0.024t(3) - 0.038t(2) - 0.044t + 0.092). The average duration of the cell cycle was 24h. Cell growth decreased as donor tissue retrieval postmortem time increased. Cultured cells featured various expressions of cytokeratin-3 ranging from absent (few cells) to strong. More than 50% of cells expressed vimentin. CONCLUSION: Limbal tissue retrieved from donor tissue that was organ-cultured for 3 weeks maintains a potential for cell renewal and growth compatible with clinical use for limbal allograft transplantation or cultured stem cell transplantation. However, cell growth potential is impaired by donor postmortem ischemia, which implies that tissue must be retrieved as soon as possible after donor death.

New test for the diagnosis of bacterial endophthalmitis.

BACKGROUND: Diagnosis of bacterial endophthalmitis (BE) often fails due to: (1) insufficient volumes of vitreous fluid (VF) and aqueous humour (AH); (2) lack of sensitivity of culture; (3) antibiotic treatments; (4) polymerase chain reaction (PCR) cross-contamination; and (5) limitations on the interpretation of the real-time PCR melting curve. We developed a fast real-time (f-real-t) PCR to improve the performance of the laboratory diagnosis of BE. METHODS: The following samples were processed after adding an internal control: phosphate buffered saline (PBS); VF, AH and cell suspensions spiked with Bacteria (Bac); VF and AH from patients with endophthalmitis; and VF and AH from non-infective patients. DNA was extracted (MagNA Pure) and added to four tubes containing selected primers and probes for the identification and quantification of all Bac and eight genera by f-real-t PCR. Diagnostic performances based on direct microscopic examination, culture and f-real-t PCR were compared. RESULTS: The f-real-t PCR detected at least 0.01 colony-forming units (CFU) of Bac/microl with no cross-reactivity with fungi. Correlation with culture-positive results was 100%. Sixty per cent of BE samples tested culture-positive, but f-real-t PCR tested positive for 90%. Samples from non-infective cases were negative. CONCLUSION: The f-real-t PCR detected and quantified Bac, Staphylococci, Streptococci, Haemophilus, Pseudomonas, Enterobacteria, Acinetobacter, Propionibacteriacae and Corynebacteria in one run. Cultures required several hours to days (with a non-negligible number of false-negative results) and the f-real-t PCR was completed in 90 min. The f-real-t PCR is presented as a new tool for the diagnosis of BE: its usefulness requires validation with larger series of samples.