Joyce Luciana Covre

Transplantation of Conjunctival Epithelial Cells Cultivated Ex Vivo in Patients With Total Limbal Stem Cell Deficiency

Purpose: To report the outcomes of transplantation of autologous conjunctival epithelial cells cultivated ex vivo (EVCAU) in patients with total limbal stem cell deficiency (LSCD). Methods: EVCAU were cultivated on denuded human amniotic membrane and transplanted in 12 eyes of 10 patients with total LSCD. We evaluated the improvement in the defined clinical parameters of LSCD (loss of corneal epithelial transparency, superficial corneal neovascularization and epithelial irregularity/recurrent epithelial breakdown), vision acuity, impression cytology, immunocytochemical analysis (CK3/CK19), and the appearance of a regular hexagonal basal layer of cells on corneal confocal microscopy. Histologic and immunohistochemical features were studied in 3 corneal buttons of patients submitted to penetrating keratoplasty after EVCAU. Results: Cultivated conjunctival epithelium formed 4 to 5 layers with the formation of basement membrane–like structures. Immunocytochemical analysis showed positivity for CK3, CK19, MUC5AC, Ki-67, P63, and ABCG2. The improvement of the clinical parameters for this treatment in our cohort was 10 of 12 (83.3%) in a mean follow-up time of 18.5 months (range, 15–26 months), and these eyes showed an improvement in impression cytology, immunocytochemistry, and in vivo confocal analysis. Corneal buttons showed a well-formed epithelium with 5 to 6 layers, with rare cells periodic acid–Schiff+, and positivity for CK3, CK19, P63, connexin 43, and MUC5AC. Conclusion: We demonstrated the preliminary results of transplantation of EVCAU for corneal surface reconstruction in cases with total LSCD. Future studies are needed to further assess the long-term efficacy of this procedure.

Comparação entre membrana amniótica com e sem epitélio como substrato para cultura de células epiteliais do limbo ex vivo

PURPOSE: To evaluate the efficacy and ultrastructural aspects of human limbal epithelial cells cultured on amniotic membrane (AM) with and without epithelium. METHODS: Limbal epithelial cell cultures were established from cadaveric cor neo-scleral rim explants derived from 6 different donors. The explants from each donor were placed under 3 different groups: on human preserved AM with epithelium (Group 1), AM deepithelialized with trypsin (Group 2) and control (Group 3). The epithelial cell migration was evaluated under phase contrast microscopy. After 15 days, the amniotic membrane with cells cultures were removed and submitted to scanning and transmission electron microscopy to check for epithelial migration and adhesion. RESULTS: All epithelial cell cultures from the controls grew over the botton of the culture plate wells until reaching confluence. Epithelial cultures grew over all but one denuded amniotic membrane. In the group amniotic membrane with epithelium, epithelial cell growing was observed only in 1 well. CONCLUSIONS: Using this model, denuded amniotic membrane appeared to be the best substrate for epithelial cell migration and adhesion comparing to amniotic membrane with epithelium. Removal of amniotic membrane epithelial seems to be an important step for establishing limbal epithelial cell culture on amniotic membrane.

Evaluation of galectin-1 and galectin-3 as prospective biomarkers in keratoconus

Aims To evaluate the expression of β-galactoside-binding proteins galectin (Gal)-1 and Gal-3 in patients with keratoconus (KC) and postcorneal collagen cross-linking (CXL) treatment in vitro. Methods Tear fluid, cornea samples and conjunctival impression cytology specimens from control and KC patients were used to evaluate Gal-1 and Gal-3 expressions. Primary keratocytes were isolated by collagenase digestion from surgically removed corneas of five normal or KC human corneal buttons and cultured in Dulbecco’s modified eagle medium/Ham’s F12 medium supplemented with 2% fetal bovine serum. These cells were evaluated under two experimental conditions: control and submitted to the application of ultraviolet A light and riboflavin 0.1% (CXL) for 30 min. Results Patients with KC displayed increased levels of Gal-1 and Gal-3 in conjunctival epithelial cells compared with control. Furthermore, KC corneas were associated with intense expression of Gal-1 in the stroma, released by keratocytes. Ultrastructural analysis of keratocytes showed a marked increase of endogenous Gal-3 levels, but not Gal-1, in KC. In vitro, CXL induced significant release of Gal-1 in keratocyte supernatants (116±18 ng/mL, P<0.05) and decreased inflammatory biomarkers as interleukin (IL)-6, IL-8, matrix metalloproteinase (MMP)-2 and MMP-9. Gal-3 levels were not detected in the keratocyte supernatants. Conclusion Gal-1 and Gal-3 represent new interesting KC biomarkers as revealed by their different expression patterns in KC and control corneal samples. CXL has an immunosuppressive effect on keratocytes by reducing the release of cytokines and MMPs and increased expression of anti-inflammatory protein Gal-1.

Variation in the volume of lubricating eyedrops available in the brazilian market

Objetivo: Avaliar a variacao intra e interexaminadores do volume de gotas dispensados de frascos de colirios lubrificantes disponiveis no mercado. Metodos: Foram estudados cinco frascos de colirios lubrificantes e dezenove voluntarios participaram deste estudo. A massa media de gotas de 20µl dos colirios foi obtida utilizando micropipeta e balanca de precisao e como padrao para comparacao com a massa das gotas obtidas pelos voluntarios. Cinco gotas de cada frasco foram pesadas individualmente com o tubo de colirio perpendicular a balanca, usando o primeiro e segundo dedos da mao direita, de forma que a pressao fosse aplicada somente no meio do frasco. Os experimentos foram realizados em uma sala climatizada a temperatura ambiente (21±1°C). Resultados: Todos os frascos de colirios apresentaram variacao estatisticamente significante das massas das gotas obtidas pelos examinadores quando comparadas com a massa media padrao de 0,0182±0,0014g, com excecao da comparacao entre os dados do colirio A com o colirio D, que nao apresentou variacao estatisticamente significante. Conclusao: O presente estudo demonstra a ausencia de uniformidade das gotas dispensadas pelos frascos de colirios disponiveis no mercado e a sua inadequacao a real necessidade, uma vez que as gotas dispensadas sao maiores do que o indicado. Esse fato torna-se um problema quando se trata de periodo de tratamento prolongado, especialmente com colirios dispendiosos como os indicados para a terapeutica do glaucoma. Nesse sentido, a padronizacao das gotas de colirios se faz necessaria.

The effects of riboflavin and ultraviolet light on keratocytes cultured in vitro

PURPOSE To culture quiescent human keratocytes and evaluate the effects of ultraviolet light and riboflavin on human corneal keratocytes in vitro. METHODS Keratocytes were obtained from remaining corneoscleral ring donor corneas previously used in corneal transplant surgeries and cultured in DMEM/F12 with 2% FBS until confluence. Characterization of cultured cells was performed by immunofluorescence analysis for anti-cytokeratin-3, anti-Thy-1, anti-α-smooth muscle actin, and anti-lumican. Immunofluorescence was performed before and after treatment of cultured cells with either ultraviolet light or riboflavin. Corneal stromal cells were covered with collagen (200 µL or 500 µL) and 0.1% riboflavin, and then exposed to ultraviolet light at 370 nm for 30 minutes. After 24 hours, cytotoxicity was determined using MTT colorimetric assays, whereas cell viability was assessed using Hoechst 33342 and propidium iodide. RESULTS Cell cultures achieved confluence in approximately 20 days. Expression of the lumican was high, whereas no expression of CK3, Thy-1, and α-SMA was observed. After crosslinking, MTT colorimetric assays demonstrated a low toxicity rate, whereas Hoechst 33342/propidium iodide staining demonstrated a low rate of apoptosis and necrosis, respectively, in all collagen-treatment groups. CONCLUSION Keratocytes can be successfully cultured in vitro and characterized by immunofluorescence using lumican. MTT colorimetric assays, and Hoechst 33342, and propidium iodide staining demonstrated a higher rate of cell death in cells cultured without collagen, indicating collagen protects keratocytes from the cytotoxic effects of ultraviolet light.