RESUMO Objetivo: Avaliar o efeito da aplicação da luz ultravioleta e riboflavina sobre ceratócitos da córnea humana in vitro. Métodos: Os ceratócitos foram obtidos a partir das rimas corneoesclerais remanescentes da trepanação de córneas previamente utilizadas em cirurgias de transplante de córnea e cultivadas em meio DMEM/F12 com 2% de FBS até atingir confluência. As culturas de células foram caracterizadas por imunofluorescência com os anticorpos K3 (marcador de células epiteliais), Thy-1 (marcador de fibroblasto) SMA (marcador de miofibroblasto) e Lumican (marcador de ceratócitos). Imunofluorescência também foi feita após o tratamento. As células do estroma da córnea foram cobertas com colágeno (200 µL e 500 µL) e 0,1% de riboflavina e exposta a luz UVA a 370 nm por 30 minutos. Após 24 horas, citotoxicidade foi determinada por ensaio de MTT e a viabilidade celular foi feita por Hoechst 33342/Iodeto de propideo. Resultados: As culturas de células atingiram confluência em aproximadamente 20 dias. Imunofluorescência apontou alta expressão para o marcador de ceratócitos (Lumican) e expressão negativa par os marcadores de células epiteliais (K3), fibroblasto (Thy-1) e miofibroblasto (α-SMA). Após o cross linking a análise de MTT mostrou baixa taxa de toxicidade e com a coloração de Hoechst 33342/Iodeto de propideo baixa taxa de apoptose e necrose respectivamente em todos os grupos que continham colágeno. Conclusão: As culturas de ceratócitos foram obtidas e caracterizadas por imunofluorescência através do marcador Lumican com sucesso. O ensaio de MTT e a coloração por Hoechst 33342 e iodeto de propídio, apresentaram maior índice de morte celular nos grupos que não continham colágeno, provando que protege as células contra os efeitos da luz UVA.

ABSTRACT 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.

Two experiments were carried out to evaluate, respectively, nitrogen balance (NB) and the productive performance of 31-to-50-day-old rabbits subjected to different levels of lysine and methionine+cystine (met+cys). Seventy-five animals were randomly distributed in 5 × 3 blocks (five levels of lysine: 5.5, 6.5, 7.5, 8.5 and 9.5 g/kg combined with three levels of met+cys: 5.0, 6.0 and 7.0 g/kg), with 15 treatments and five replications for the NB assay. The assay lasted 14 days: 10 days for acclimatization and four days for feces and urine collection. Increasing met+cys levels had a quadratic effect on the nitrogen excreted in urine (NU): the lowest excretion was found at the dietary level of 5.9 g/kg met+cys. Increasing lysine levels also affected NU and nitrogen retained daily (NR): the lowest NU was obtained at the dietary level of 7.28 g/kg lysine, and maximum NR was found at 7.24 g/kg lysine. Increases in met+cys levels in the diets affected neither performance nor carcass characteristics of rabbits up to 50 days of age. On the other hand, body weight at 50 days, daily weight gain and feed conversion of rabbits slaughtered at 50 days had a quadratic effect as the lysine levels increased. The best results were found at 7.5, 7.38 and 7.36 g/kg lysine. Lysine and met+cys levels of 7.4 and 5.0 g/kg in the diet are recommended for 31-to-50-day-old rabbits.

The objectives of this study were to determine the chemical composition of vegetable and mixed crude glycerin and digestible energy to and evaluate different inclusion levels in rabbit feeding. A total of 108 rabbits were used in the digestibility assay, assigned in a completely randomized design into nine treatments, one reference diet plus eight test diets, where the glycerin types were included at levels of 4, 8, 12 and 16%, replacing the reference diet roughage. In the performance assay, 180 rabbits were assigned to a completely randomized design into a 2 × 4 factorial arrangement (two types of glycerin and four levels of inclusion: 3, 6, 9 and 12%) plus a reference diet and ten replications. The vegetable and mixed crude glycerin showed digestible energy of 5,099 and 4,953 kcal/kg of dry matter, respectively. Although feed intake decreased linearly with the growing inclusion of both kinds of glycerin during both evaluated periods (from 32 to 50 and 32 to 70 days), performance losses occurred only with the inclusion of vegetable crude glycerin. The inclusion of mixed crude glycerin up to 12% allowed similar animal performance to the reference diet up to 50 days, unlike vegetable crude glycerin, for which inclusions over 6% in the diet resulted in poorer performance compared with that obtained with the reference diet. In the total period, differences were not observed for live weight or weight gain of the animals fed both glycerin types. Carcass weight decreased linearly with the inclusion of both glycerin types, with lower values compared with the reference diet only for the two highest levels of glycerin inclusion. Mixed crude glycerin can be included up to the maximum level studied (12%), while vegetable glycerin can be added up to 6% in the diet of growing rabbits.