Ocular surface findings in impression cytology after interferon a2b or mitomycin C in rabbits / Achados em citologia de impressão da superfície ocular após uso de interferon alfa-2b ou mitomicina C: estudo em coelho

ABSTRACT Objective: To describe ocular surface findings in impression cytology obtained from healthy rabbit conjunctiva treated with interferon alpha-2b eyedrop, and compare them to findings after use of mitomycin C 0.02%. Methods: An experimental study using a rabbit model was performed between September 2013 and October 2014 at the Faculdade de Medicina de Marília, Universidade Federal de São Paulo, Clínica de Olhos Moacir Cunha. Thirty New Zealand white rabbits were divided into 6 groups and received interferon alpha-2b or mitomycin C 0.02%. Impression cytology (IC) was performed prior to topical applications and at15, 30 and 60 days of use. The following variables were analyzed in impression cytology: goblet cells, cellularity, cell-to-cell adhesion, nucleus/cytoplasm ratio, nuclear chromatin, inflammatory cells keratinization, and cytomegaly. Results: The major findings in impression cytology after us of interferon alpha-2b included loss of goblet cells (50.8%), reduced cell-to-cell adhesion (26.2%), abnormal nucleus/cytoplasm ratio (20%) and reduced cellularity (15.4%). After use of mitomycin C 0.02%, the most common changes included loss of goblet cells (46.2%), abnormal nucleus/cytoplasm ratio (25.6%), less cell-to-cell adhesion (23.1%), and reduced cellularity (20.5%). There were no significant differences in any variable when comparing impression cytology after interferon alpha-2b and after mitomycin C 0.02%. Goblet cell loss was more pronounced at days 30 and 60, as compared to impression cytology at day 15 for both drugs. Conclusion: The loss of goblet cells, reduced cell-to-cell adhesion and cellularity, along with abnormal nucleus/cytoplasm ratio were the most common findings in impression cytology after use of interferon alpha-2b. These findings are similar to those described for use of mitomycin C 0.02%. ..

RESUMO Objetivo: Descrever os achados em citologia de impressão de conjuntiva sadia de coelho submetida ao uso de colírio de interferon alfa-2b e compará-los ao que foi encontrado após uso da mitomicina C 0,02%. Métodos: Estudo experimental realizado em modelo animal no período entre setembro de 2013 e outubro de 2014 nas dependências da Faculdade de Medicina de Marília, da Universidade Federal de São Paulo e da Clínica de Olhos Moacir Cunha. Trinta coelhos albinos da raça Nova Zelândia foram divididos em seis grupos e receberam interferon alfa-2b ou mitomicina C. A citologia de impressão foi realizada antes do início dos colírios e após 15, 30, 60 dias de seu uso. As seguintes variáveis foram analisadas na citologia de impressão: células caliciformes, celularidade, adesão intercelular, razão núcleo/citoplasma, cromatina, células inflamatórias, queratinização e citomegalia. Resultados: Os principais achados na citologia de impressão após o uso do interferon alfa-2b foram a redução de células caliciformes (50,8%), a diminuição da adesão intercelular (26,2%), a alteração da razão N/C (20%) e a redução da celularidade (15,4%). Após o uso da mitomicina C 0,02%, foram mais frequentes a redução das células caliciformes (46,2%), a alteração da razão N/C (25,6%), a adesão intercelular (23,1%) e a redução da celularidade (20,5%). Não houve diferença estatisticamente significante para nenhuma das variáveis estudas quando se compararam as citologias de impressão após interferon alfa-2b com as citologias de impressão após mitomicina C 0,02%. Independentemente da substância utilizada, as citologias colhidas 30 e 60 dias após início das drogas apresentaram maior redução de células caliciformes quando comparadas com as citologias de impressão colhidas após 15 dias. Conclusão: A redução das células caliciformes, a diminuição da adesão intercelular, a alteração da razão N/C e a diminuição da celularidade foram as alterações mais frequentes na citologia de impressão colhida após o uso de interferon alfa-2b. Os achados em citologias de impressão após o uso de interferon alfa-2b são semelhantes àqueles encontrados após o uso da mitomicina C 0,02%.

Evaluation of Stem Cell Components in Retrocorneal Membranes

The purpose of this study was to elucidate the origin and cellular composition of retrocorneal membranes (RCMs) associated with chemical burns using immunohistochemical staining for primitive cell markers. Six cases of RCMs were collected during penetrating keratoplasty. We examined RCMs with hematoxylin and eosin (H&E), periodic acid-Schiff (PAS) staining and immunohistochemical analysis using monoclonal antibodies against hematopoietic stem cells (CD34, CD133, c-kit), mesenchymal stem cells (beta-1-integrin, TGF-beta, vimentin, hSTRO-1), fibroblasts (FGF-beta, alpha-smooth muscle actin), and corneal endothelial cells (type IV collagen, CD133, VEGF, VEGFR1). Histologic analysis of RCMs revealed an organized assembly of spindle-shaped cells, pigment-laden cells, and thin collagenous matrix structures. RCMs were positive for markers of mesenchymal stem cells including beta-1-integrin, TGF-beta, vimentin, and hSTRO-1. Fibroblast markers were also positive, including FGF-beta and alpha-smooth muscle actin (SMA). In contrast, immunohistochemical staining was negative for hematopoietic stem cell markers including CD34, CD133 and c-kit as well as corneal endothelial cell markers such as type IV collagen, CD133 except VEGF and VEGFR1. Pigment-laden cells did not stain with any antibodies. The results of this study suggest that RCMs consist of a thin collagen matrix and fibroblast-like cells and may be a possible neogenetic structure produced from a lineage of bone marrow-derived mesenchymal stem cells.

Tempo de reepitelização corneana com a instilação de colírio contendo hialuronato de sódio e carboximetilcelulose / Corneal reepithelialization time with instillation of eye drops containing sodium hyaluronate and carboxymethylcellulose

OBJETIVO: Avaliar o tempo de reepitelização corneana pós abrasão usando colírios comercialmente disponíveis, um contendo hialuronato de sódio a 0,4%, outro contendo carboximetilcelulose a 1%, e comparar com a reepitelização sem instilação de colírio. MÉTODOS: Foram utilizados 24 coelhos, nos quais foi feita a abrasão mecânica da córnea nos 8 mm centrais. Esses animais foram divididos em três grupos. O primeiro grupo recebeu um colírio disponível comercialmente contendo hialuronato de sódio 0,4%, o segundo recebeu um colírio contendo carboximetilcelulose 1% e o terceiro não recebeu nenhuma droga. A avaliação foi feita a cada 24 horas por meio da análise de fotografias digitais sob luz azul de cobalto e coramento das córneas com fluoresceína a 2%. O estudo das imagens foi feito pelo sistema de análise de imagens do Autocad 2009®. A análise dos dados foi feita comparando o tempo total de reepitelização da córnea e a cada 24 horas entre os três grupos. RESULTADOS: A velocidade de reepitelização do grupo que usou colírio contendo hialuronato de sódio foi em média 90 horas; o grupo que usou carboximetilcelulose apresentou média de 105 horas; e o grupo que não usou nenhum tipo de lubrificante apresentou média de 108 horas para total reepitelização. Houve uma melhor performance na reepitelização após 96 horas nas córneas dos coelhos que usaram os colírios lubrificantes, sendo essa diferença estatisticamente comprovada. CONCLUSÃO: O colírio contendo hialuronato de sódio 0,4% mostrou índice de eficácia maior que aquele contendo carboximetilcelulose 1%, e este maior eficácia que o controle. Os resultados encontrados neste estudo mostram que o uso de lubrificantes no processo de reepitelização são de extrema valia e devem ser usados de rotina na clínica oftalmológica.

PURPOSE: Evaluate the time of post-abrasion corneal re-epithelialization using commercially available eye drops, one of which containing 0.4% sodium hialuronate, and the other containing 1% carboxymethylcellulose, and compare them to the re-epithelialization without the drops. METHODS: 24 rabbits were used, which had the mechanical abrasion of the central 8 mm of their corneas done. These animals were divided in 3 groups. The first one received the drops containing 0.4% of sodium hialuronate, the second one received the drops containing 1% of carboxymethylcellulose and the third group did not receive any drugs. The evaluations took place every 24 hours through the analysis of digital pictures under cobalt blue light and coloring of the corneas with 2% fluorescein. The pictures were analyzed with the software Autocad 2009®. The data was analyzed through the comparison of the total re-epithelialization time among the three groups RESULTS: The time of total re-epithelialization of the group using sodium hialuronate was on average 90 hours and the group using carboxymethylcellulose 105 hours, while the group using no drugs was 108 hours. There was a better performance of those groups using the drops and this difference can be proved statistically. CONCLUSION: The drops containing 0.4% of sodium hialuronate showed a higher efficiency rate compared to the drops containing 1% of carboxymethylcellulose, which was higher than the control group. The results of the present study show that the use of lubricants in the process of re-epithelialization are extremely valid and must be used frequently in ophthalmologic clinic.

Motivos do descarte de córneas captadas pelo banco de olhos do Hospital São Paulo em dois anos / Discarding causes of cornea collected by eye bank of São Paulo hospital in two years

OBJETIVO: Analisar os motivos do descarte ou não utilização de córneas doadas, cujos tecidos foram captados, preservados e avaliados pelo Banco de Olhos do Hospital São Paulo no período de outubro de 2002 a setembro de 2004. MÉTODOS: Estudo retrospectivo analisando os prontuários com as seguintes informações sobre as doações: história clínica e ocular do doador, idade, causa mortis, resultados de exames sorológicos, avaliação do corpo do doador, avaliação biomicroscópica da morfologia do globo ocular e da córnea e contagem celular endotelial. RESULTADOS: De 1.116 córneas doadas no período, 518 (46,41 por cento) foram descartadas antes de serem preservadas, sendo 288 (25,80 por cento) devido à causa mortis, 56 (5,01 por cento) pelo histórico clínico do doador e 174 (15,59 por cento) pela avaliação do tecido. Das 598 córneas preservadas, 317 (28,40 por cento) foram destinadas ao transplante óptico e 168 (15,05 por cento) indicadas para transplantes tectônicos. CONCLUSÃO: A análise do prontuário hospitalar e história clínica do doador foi um importante fator de descarte de córneas antes da preservação. Já a avaliação biomicroscópica realizada na lâmpada de fenda é uma etapa fundamental para qualificação e classificação do tecido adequando-o para o paciente e garantindo um bom resultado cirúrgico.

PURPOSE: To analyze causes of discarded donor corneas which were collected and preserved by São Paulo Hospital Eye Bank in a period of two years. METHODS: Retrospective study based in the investigation of donor's medical records, for clinical and ocular factors, age, death cause, serology results, body conditions, slit lamp corneal examination and endothelial cells counting. RESULTS: 1,116 corneas were donated and included in the study, 518 (46.41 percent) were discarded before preservation, 168 (15.05 percent) were not transplanted for optical purpose, but used for tectonic indication. The main reason of cornea discard was the cause of death in 288 (25.80 percent) corneas, second the slit lamp analysis of tissues in 174 (15.59 percent) corneas. CONCLUSION: The analysis of the medical records and the donor's clinical history was an important factor of cornea discard of the not preserved tissues, showing the importance of this stage to guarantee the quality of the tissue and to ensure proper safety for the recipient. Also, the biomicroscopy evaluation realized in the slit lamp is a fundamental stage for qualification and classification of the tissue to guarantee a good surgical result.

Putative stem cell markers in limbal epithelial cells cultured on intact & denuded human amniotic membrane.

BACKGROUND & OBJECTIVES: The ocular surface is an ideal region to study the epithelial stem cell (SC) biology because of the unique spatial arrangement of stem cells and transient amplifying cells. A major challenge in corneal SC biology is the ability to identify SC in vitro and in situ, and one of the major controversies in the field relates to reliable SC markers. This study was carried out to evaluate and compare the expression of the stem cell associated marker: ABCG2, keratinocyte stem cell marker: p63 and corneal differentiation markers: Cnx43 and K3/K12 on limbal explants cultured on human amniotic membrane (HAM) with intact epithelium and HAM denuded of its epithelium. METHODS: Human limbal biopsies obtained from the cadaveric donor eyes were used in this study. The cells were cultured over the HAM with intact and denuded epithelium. Reverse transcriptase PCR, immunohistochemistry, Western blotting for ABCG2, P63, Cnx43 and K3/K12 were done. RESULTS: The limbal epithelial cells cultured over intact HAM expressed the stem cell associated markers (ABCG2, p63) and showed reduced expression of the differentiation markers (Cnx43 and K3/K12) when compared to limbal epithelial cells cultured over denuded HAM, which expressed more differentiation markers at the end of three weeks. BrdU label retaining cells were observed in the limbal epithelial cells cultured over HAM with epihelium only. INTERPRETATION & CONCLUSIONS: Our results showed that the intact HAM supported the growth of limbal epithelial cells expressing stem cell associated markers, and allowing little differentiation of the limbal cells to cornea phenotype. Further studies are needed to understand the properties of the amniotic epithelium that retains the stemness in the cultured limbal stem cells.

Corneal endothelial cell loss between the Kongsap manual phacofragmentation and phacoemulsification.

OBJECTIVE: To compare corneal endothelial cell loss between the Kongsap manual phacofragmentation and phacoemulsification. MATERIAL AND METHOD: One hundred two eyes with age-related cataract were randomized to undergo either the Kongsap manual phacofragmentation (Group 1, 52 eyes) or phacoemulsification surgery (Group 2, 50 eyes) with implantation of a posterior chamber, foldable, acrylic intraocular lens performed by one surgeon. The main parameters were corneal endothelial cell density (ECD), best corrected visual acuity (BCVA), and intraoperative and postoperative complications. Follow-up visits were scheduled at 1, 4, and 12 weeks. RESULTS: Pre-operatively, the mean ECD in Group 1 was 2,350 +/- 229 cells/mm2 and in Group 2 was 2,429 +/- 263 cells/mm2 (p = 0.112). Mean ECD decrease was 7.61% in Group 1 and 7.19% in Group 2 at the end of 12 weeks. The 95% confidence intervals of the mean differences of the endothelial cell loss at 4 weeks and 12 weeks after surgery were -1.87 to 2.04% and -2.77 to 3.63%, respectively. Mean best-corrected visual acuity at the end of 4 weeks was 0.88 +/- 0.22 in Group 1 and 0.82 +/- 0.24 in Group 2 (p = 0.117). There was no statistical difference between the groups in intra-operative and postoperative complications (p > 0.05). CONCLUSION: The corneal endothelial cell loss after cataract surgery with the Kongsap manual phacofragmentation is equivalent to those of phacoemulsification and both surgical techniques allowed excellent visual results.

Effect of p27Kip1 inhibition on proliferation of bovine corneal endothelial cells by RNA interference / 华中科技大学学报（医学）（英德文版）

Three plasmids (pGenesil-P1, pGenesil-P2, pGenesil-P3) with different p27Kip1-shRNA sequences were designed and synthesized. Their effects on the proliferation of bovine corneal endothelial cells (bCEC) were investigated. Plasmid expressing irrelevant shRNA with a random combination was used as negative control (pGenesil-HK). The recombination of four plamids was confirmed by restrictive enzyme digestion and sequence analysis. The expression of mRNA and protein of p27Kip1 was detected by RT-PCR and Western blotting after stable transfection. The expressions of p27Kip1 mRNA and p27Kip1 protein of pGenesil-P1 group, pGenesil-P2 group and pGenesil-P3 group were all lower than those in the pGenesil-HK group and the blank group (non-transfected group). pGenesil-P3 had the strongest inhibitory effect and was selected for the next steps. The proliferation rates of the pGenesil-P3 group, the pGenesil-HK group and the blank group were assessed by MTT. The influence of shRNA-p27Kip1 on bCEC cell cycle was detected by flow cytometry (FCM). Compared with the control groups, the proliferation rate of the pGenesil-P3 group was increased significantly, and the ratio of S-phase also increased. It is concluded that shRNA-p27Kip1 could down-regulate the expression of p27Kip1 effectively and increase the proliferation of bCEC. RNA interference (RNAi) may be an effective means to promote the proliferation of CEC.

Avaliação da cinética do epitélio corneano com marcadores de proliferação celular / Evaluation of the corneal epithelium kinetics using cell proliferation markers

OBJETIVO: Avaliar a cinética celular do epitélio corneano de coelhas em três situações: controle, hiperproliferação e hipoproliferação celular, com a utilização dos marcadores de proliferação celular BrdU, Ki-67/MIB-1 e AgNOR. MÉTODOS: Foram utilizadas quinze coelhas albinas que tiveram seus olhos aleatoriamente divididos em 3 grupos (A, B e C). O grupo A incluiu olhos que foram submetidos à instilação de tampão fosfato (total de 10 olhos); o grupo B, instilação de tampão fosfato após a remoção de uma área central do epitélio corneano de 10 mm (total de 10 olhos) e o grupo C, instilação de 5-fluoruracil em superfície ocular íntegra (total de 10 olhos). RESULTADOS: Os resultados da média e desvio-padrão do número de células marcadas pela BrdU nos grupos A, B e C foram, respectivamente, de 7,17 ± 0,74; 35,00 ± 3,01 e 0,22 ± 0,1 células marcadas por 100 células basais. As diferenças entre os grupos foram estatisticamente significantes. A média e o desvio padrão do número de células marcadas utilizando o Ki-67 foram de 7,55 ± 1,22 no grupo A; 35,55 ± 3,84 no grupo B e 0,34 ± 0,14 no grupo C. As diferenças entre os grupos foram estatisticamente significantes. A média e o desvio-padrão da medida da área das NORs no grupo A foram de 1,92 ± 0,24, no grupo B foram de 3,61 ± 0,27 e no grupo C foram de 1,71 ± 0,26. CONCLUSÕES: Os marcadores BrdU, Ki-67 e AgNOR apresentaram uma correlação positiva e estatisticamente significante nas situações de proliferação celular avaliadas (controle, hiperproliferação e hipoproliferação); o emprego do AgNOR não permitiu identificar diferenças na proliferação celular nas situações controle e hipoproliferação e houve maior concordância de resultados entre a BrdU e o Ki-67 nas três situações de proliferação celular.

PURPOSE: In order to maintain its clear and uniform structure, the corneal epithelium needs constant equilibrium between production (division) and desquamation of its epithelial cells. The author aimed to evaluate the cell kinetics of corneal epithelium of rabbits in three situations (control, hypoproliferation and hyperproliferation) using BrdU, Ki-67/MIB-1 and AgNORs proliferation markers. METHODS: Fifteen white female rabbits had their eyes randomly divided into three groups (A, B and. C). Group A included eyes submitted to phosphate buffer saline instillation (total 10 eyes); group B, instillation of phosphate buffer saline after removing a 10 mm central area of the corneal epithelium (total 10 eyes) and group C, instillation of 5-fluorouracil in normal epithelium (total 10 eyes). RESULTS: The results of the mean number and standard deviation of the marked cells using BrdU in groups A, B e C were, respectively, 7.17 ± 0.74; 35.00 ± 3.01 e 0.22 ± 0.1 marked cells per 100 basal cells. Differences among groups were statistically significant. The mean number and standard deviation of the labelled cells using Ki-67 were 7.55 ± 1.22 in group A; 35.55 ± 3.84 in group B and 0.34 ± 0.14 in group C. Differences among groups were statistically significant. The mean area and standard deviation of NORs in group A were 1.92 ± 0.24, in group B, 3.61 ± 0.27 and in group C, 1.71 ± 0.26. CONCLUSIONS: The markers BrdU, Ki-67 and AgNOR showed a positive correlation with statistical significance among the cellular proliferation situations studied (control, hypoproliferation and hyperproliferation); the AgNOR did not show statistically significant differences among the control and hypoproliferation situations and there was more agreement in the results among markers BrdU and Ki-67 in three cell proliferation situations.

Avaliação de córneas doadoras em lâmpada de fenda e microscopia especular durante o período de armazenamento / Evaluation of corneal tissue by slit lamp and specular microscopy during the preservation period

OBJETIVO: Analisar as mudanças de notas de avaliação por meio da biomicroscopia e microscopia eletrônica de córneas doadas, e desta forma assegurar córnea doadora de padrão satisfatório ao paciente. MÉTODOS: Foram avaliadas prospectivamente 203 córneas entre setembro de 2002 e fevereiro de 2003, doadas ao Banco de Olhos de Sorocaba. A avaliação constava de graduação de 0 a 3 que variava mediante os seguintes fatores: exposição e defeito epitelial, opacidade e infiltrado estromal, dobras ou estrias de Descemet, pleomorfismo, polimegatismo e guttata, "endothelial snail track", edema, refletividade. Os dados encontrados foram correlacionados com o intervalo entre óbito e preservação, contagem de células endoteliais e idade dos doadores. RESULTADOS: Duzentos e três córneas foram avaliadas. A idade média dos doadores foi de 55 anos (dp=14,8 anos). O tempo médio do óbito à preservação foi de 9,1 h (mínimo de 2 horas e máximo de 25 horas). Oitenta e seis córneas sofreram pioras de avaliação, sendo que 66,3 por cento delas tinham até 2.500 células endoteliais e 59,3 por cento delas tinham tempo superior a 6 horas entre o óbito e a preservação. O dia da mudança teve por mediana 5. CONCLUSÕES: Córneas de doadores mais jovens possuem significativamente avaliações melhores que doadores mais velhos. O tempo médio de mudança de avaliação foi com 5 dias em 50 por cento das córneas, entretanto, expressiva parcela de córneas apresentou alteração nos dias 6°, 7° e 8°. Córneas preservadas após 6 horas do óbito têm maior tendência à perda de células e redução dos índices de avaliação.

PURPOSE: To analyze changes evaluation of corneal grafts by slit lamp and electron microscopy examination in order to ensure a donor cornea of good quality level for the patient. METHODS: 203 córneas donated to the Sorocaba Eye Bank between September 2002 and February 2003 were prospectively evaluated. The evaluation was graded from 0 to 3 according to the following factors: exposure and epithelial damage, stromal opacity, Descemet folds, pleomorphism, polymegatism and guttata, endothelial snail track, edema, reflectivity. The data were correlated with time between death and preservation, endothelial cell count and donor's age. RESULTS: 203 corneas were evaluated. The mean age of donors was 55 years (dp= 14.8 years). The mean time between death and preservation was 9.1 h (minimum of 2 h and maximum of 25 h). Eighty-six corneas suffered worsening of evaluation, whereby 66.3 percent had less than 2,500 endothelial cell count and 59.3 percent presented time between death and preservation over 6 hours. The mean day of the grading change was the 5th. CONCLUSIONS: Corneal grafts from younger donors had significantly better evaluation than those of older donors. The mean time of the evaluation change was on the 5th day in 50 percent of the corneas, however, an expressive number of corneas suffered changes on the 6th, 7th and 8th day. Corneal graft preserved after 6 hours of death had a greater tendency to lower cell count and to decrease in evaluation grades.

Comparison of specific activity and cytopathic effects of purified 33 kDa serine proteinase from Acanthamoeba strains with different degree of virulence

The pathogenic mechanism of granulomatous amebic encephalitis (GAE) and amebic keratitis (AK) by Acanthamoeba has yet to be clarified. Protease has been recognized to play an important role in the pathogenesis of GAE and AK. In the present study, we have compared specific activity and cytopathic effects (CPE) of purified 33 kDa serine proteinases from Acanthamoeba strains with different degree of virulence (A. healyi OC-3A, A. lugdunensis KA/E2, and A. castellanii Neff). Trophozoites of the 3 strains revealed different degrees of CPE on human corneal epithelial (HCE) cells. The effect was remarkably reduced by adding phenylmethylsulfonylfluoride (PMSF), a serine proteinase inhibitor. This result indicated that PMSF-susceptible proteinase is the main component causing cytopathy to HCE cells by Acanthamoeba. The purified 33 kDa serine proteinase showed strong activity toward HCE cells and extracellular matrix proteins. The purified proteinase from OC-3A, the most virulent strain, demonstrated the highest enzyme activity compared to KA/E2, an ocular isolate, and Neff, a soil isolate. Polyclonal antibodies against the purified 33 kDa serine proteinase inhibit almost completely the proteolytic activity of culture supernatant of Acanthamoeba. In line with these results, the 33 kDa serine proteinase is suggested to play an important role in pathogenesis and to be the main component of virulence factor of Acanthamoeba.

Deep stromal mycobacterial keratitis: viable bacteria after six months of treatment: case report and literature review

O objetivo do caso é descrever a presença de micobactérias viáveis em pacientes com ceratite, 6 meses após tratamento intensivo. A identificação de espécies, foi efetuada usando método PRA (polymerase chain reaction seguida pela restriction endonuclease analysis). Clonalidade foi avaliada pelos métodos RAPD (randomly amplified polymorphic DNA) e ERIC-PCR (enterobacterial repetitive intergenic consensus - polymerase chain reaction). Paciente refere trauma com corpo estranho metálico há 3 semanas. A cultura da córnea revelou Mycobacterium abscessus. Após 6 meses de tratamento tópico e sistêmico, paciente apresentava-se sem inflamação, sendo considerado clinicamente curado. Realizou-se então, uma ceratoplastia penetrante com intuitos ópticos. A cultura da córnea transplantada revelou micobactérias de mesma origem clonal. O achado mais interessante neste relato, foi a positividade da cultura da córnea transplantada após 6 meses de intenso tratamento específico. Ao nosso conhecimento, esse é o primeiro caso relatado na literatura mostrando essa possibilidade em tratamento de ceratites por micobactérias. Assim, os pacientes com ceratite por Mycobacterium abscessus podem apresentar bactérias viáveis após longo tempo de tratamento específico e precisam ser seguidos cuidadosamente por um longo período de tempo.

Cationic liposome-mediated bcl-xl gene transfection into human keratocytes / 华中科技大学学报（医学）（英德文版）

The efficiency and safe range of Lipofectamine2000 (LF2000)/bcl-xl applied in human keratocytes, the optimal ratio of LF2000/bcl-xl and the bcl-xl gene expression in human keratocytes were investiaged. By using trypan-blue staining, the effects of LF2000 and bcl-xl on the survival rate of the cultured human keratocytes were measured respectively. By using semi-quantitative RT-PCR, the efficiency and the expression of LF2000-mediated bcl-xl transfection into keratocytes were examined. The results showed that the survival rate of human keratocytes had no signficant change in the presence of LF2000 (20 microg/ml) or bcl-xl (10 microg/ml) for 24 h. LF2000 could effectively mediate the transfection of exogenous gene bcl-xl into human keratocytes. The best transfection efficiency could be obtained when the ratio of bcl-xl/LF2000 was 1:8. One day after transfection, the positive cells for bcl-xl could be detectable, and the positive rate reached the peak-on the posttransfection day 3 (48.3%), then gradually decreased. Fifteen days after transfection, there were few positive cells. It was suggested that LF2000 could effectively transfer the exogenous gene bcl-xl into human keratocytes without obvious toxicity during a concentration range. LF2000/bcl-xl may be likely to play an important role in gene therapy of human keratocytes.

Protective Effect of Heat Shock Protein 70 Against Oxidative Stresses in Human Corneal Fibroblasts

We evaluated DNA protection effect of heat shock protein (HSP) against cytotoxic effects of exogenous nitric oxide (NO) and reactive oxygen intermediate (ROI). Cultured human corneal fibroblasts were divided into 4 groups. Control (Group I) was not exposed to a sub-lethal heat treatment. Other 3 groups were exposed to 43 degrees C for 1 hr, then incubated at 37 degrees C during different duration (1, 6, 24 hr, Group II, III, IV, respectively). Expression pattern of HSP 70 was analyzed by Western blot. Cell viability was measured by MTT assay and the relationship between HSP 70 expression and DNA damage was examined by terminal deoxyribonucleotidyl transferase mediated dUTP-digoxigenin nick and labeling (TUNEL) stain and single cell gel electrophoresis. Expression pattern of HSP 70 was dependent on recovery times. Cell viability following heat treatment was significantly increased and the TUNEL positive cell number was decreased at 6 hr. In single cell gel electrophoresis, tail moments were increased in a dose-dependent manner by SNAP and X/XO. Following heat treatment, tail moments showed decreased significantly at 6 hr. These results suggest that induction of HSP 70 by sub-lethal heat treatment is closely related with cytoprotective effects against oxidative stresses in human corneal fibroblasts.

In-vivo slit scanning confocal microscopy of normal corneas in Indian eyes.

OBJECTIVE: To study the cellular populations of healthy corneas of Indian eyes using confocal microscopy and to evaluate the correlation with age, gender and laterality. METHODS: The central corneas of 100 eyes of 50 healthy subjects were examined using an in-vivo slit scanning confocal microscope (Confoscan 2). Images were analysed for cell densities of the epithelium, stroma and endothelium. RESULTS: Good quality images enabling analysis of all cell layer populations were obtained in 74 eyes of 43 healthy subjects (22 males and 21 females) with a mean age of 31.89 +/- 13.47 (range 19-71 years). The basal epithelial cell density was 3601.38 +/- 408.19 cells/mm2 (range 3017.3-4231.1 cells/mm2). The mean keratocyte nuclei density in the anterior stroma was 1005.02 +/- 396.86 cells/mm2 (range 571.6-1249.6 cells/mm2) and in the posterior stroma was 654.32 +/- 147.09 cells/mm2 (range 402.6-1049.1 cells/mm2). Posterior keratocyte nuclei density was 30.76% less than the anterior stromal keratocyte nuclei density. The difference in keratocyte nuclei density was statistically significant (P=0.001). The mean endothelial cell density was 2818.1 +/- 361.03 cells/mm2 (range 2118.9-4434 cells/mm2) and the mean endothelial cell area was found to be 385.44 +/- 42.66 mm2 (range 268.9-489.2 mm2). Hexagonal cells formed 22.5-69.4% of the endothelial cell populations (mean 42.04 +/- 11.81%). Mean coefficient of cell size variation was 32.29 +/- 3.06 (range 27.2-39.2). No statistically significant differences were found in cell densities of any corneal layer either between female and male patients or between right and left eyes. Basal epithelial cell density, anterior stromal keratocyte nuclei and posterior stromal keratocyte nuclei density were unaffected by age (r=0.12, 0.07, -0.12 respectively) (P=0.001). There was a statistically significant negative correlation between mean endothelial cell density and increase in age (r=-0.42, P=0.001). Coefficient of cell size variation and age were positively correlated (r=0.73, P=0.001). CONCLUSION: In-vivo slit scanning confocal microscopy is useful for the study of corneal cell populations. Our study provides normative data of these cell populations.

Citologia de impressäo de superficíe ocular: técnica de exame e de coloraçäo / Impression cytology of the ocular surface: examination technique and staining

Objetivo: Apresentar uma técnica de exame e de coloraçäo de amostras de citologia de impressäo da superfície ocular desenvolvida em serviço de referência. Método: Obtiveram-se 28 amostras de citologia de impressäo de pacientes com alteraçöes da superfície ocular no Setor de Doenças Externas Oculares no período de julho a novembro de 1999. Coraram-se e avaliaram-se as amostras microscopicamente no Laboratório de Microbiologia Ocular, do Departamento de Oftalmologia da Universidade Federal de Säo Paulo - Escola Paulista Medicina. Resultados: Desenvolveu-se um modelo de papel de filtro com ápice, base e abertura lateral, que forneceu seu posicionamento correto no olho no momento da colheita e na lâmina para afixaçäo e coloraçäo. A técnica de coloraçäo descrita, que usa ácido períódico-Schiff, hematoxilina e Papanicolaou, é um procedimento econômico e fácil, cora as células caliciformes de róseo e as epiteliais de roxo. Conclusöes: A técnica de exame mostrou-se ideal na avaliaçäo celular das amostras de citologia de impressäo. A citologia de impressäo é um método bastante confiável para o estudo da superfície ocular, no acompanhamento da evoluçäo de patologias externas, e provou ser um procedimento realmente simples, mais barato e mais confortável para o paciente que as biópsias invasivas.

Comparison of optical and ultrasound pachometry.

PURPOSE: (1) To determine the agreement between optical and ultrasound pachometry for central corneal thickness measurements used to "correct" applanation intraocular pressure (IOP) readings. (2) To determine the inter- and intra-observer variability of optical and ultrasound pachometry. METHOD: Central corneal thickness (CCT) was measured in a masked manner using optical and ultrasound pachometry in 50 normal eyes. To assess intra- and inter-observer variability, multiple masked measurements were obtained in 51 eyes (optical pachometry) and 34 eyes (ultrasound pachometry). Agreement was determined by a published technique that uses the mean of the differences, standard error (SE) and standard deviation (SD). RESULTS: The mean difference in CCT between optical and ultrasound pachometry was 0.001 mm (SD 0.031 mm; SE 0.00439 mm). The mean inter-observer difference for the optical pachometer was 0.019 mm (SD 0.049 mm; SE 0.0069); the mean intra-observer difference was 0.003 mm (SD 0.017; SE 0.0.0024). The mean inter-observer difference for ultrasound pachometry was 0.001 mm (SD 0.009; SE 0.0015) and the mean intra-observer difference was 0.002 mm (SD 0.011; SE 0.0019). CONCLUSIONS: Ultrasound pachometry is the more reliable method for the measurement of central corneal thickness used to correct applanation IOP values. Optical pachometry had good intra-observer variability. The range of error in IOP correction for corneal thickness (inter-observer) that can occur using the ultrasound pachometer is -1.2 mmHg to +1.4 mmHg as compared to -5.6 mmHg to +8.5 mmHg with the optical pachometer.

Observations on iris melanocytes implanted in the cornea

The pattern and morphology of cellular infiltration of iris melanocytes implanted into the corneal stroma were studied with a rabbit corneal model. Iris melanocytes are transformed into fibroblast-like cells with a loss of pigment granules, which may reflect the in vivo characteristics of iris melanocytes under pathologic conditions. The metaplastic chararter of iris melanocytes appears to be related to the formation of retrocorneal pigmentation and fibrous membrane.

Açao da metilcelulose a 2 por cento na câmara anterior do olho do coelho: estudo biomicroscópico, paquimétrico, tonométrico, citológico e histológico / Action of methylcelulose a 2 per cent in the anterior chamber of the eye of rabbit: study biomicroscopic, pachimetric, cytologic and histologic

Utilizaram-se 45 coelhos divididos em dois grupos: o Grupo Po (22 coelhos) e o Grupo Pk (23 coelhos). Todos sofreram substituição parcial do humor aquoso por soluções de Metilcelulose a 2 por cento em solução salina balanceada (SSB) em um olho, e SSB no olho contralateral. No grupo Po foram estudados os comportamentos do diâmetro pupilar, biomicroscópico e tonométrico, antes e depois (2, 4, 8h e 1§, 2§, 3§, 4§, 5§, 10§ e 20§ dia) das injeções das soluções na câmara anterior. No Grupo Pk foi observado o comportamento da espessura corneana antes e depois (4 e 18h) da injeção das soluções na câmara anterior, sendo em seguida realizada a histologia plana do endotélio corneano para avaliação do padrão e densidade celular. Foi colhido humor aquoso de 10 pares de olhos desse grupo para coloração e estudo. Ainda foram selecionados alguns olhos do grupo teste e controle para estudo histológico. Os resultados mostraram que nem a Metilcelulose nem a solução salina balanceada influenciaram no diâmetro pupilar. A pressão ocular dos olhos injetados com Metilcelulose aumentou em média 13,68 mmHg em relação ao olho-controle, porém com grande variabilidade de valores. O pico de pressão ocorreu às 2h, retornando aos níveis basais em 24 h. A biomicroscopia, a citologia e a histologia mostraram que a substância com um maior número de células no humor aquoso, além de uma irite localizada em 36,3 por cento dos casos. A Metilcelulose tende a se condensar e distribuir-se em diferentes locais da câmara anterior antes de desaparecer. Não foi evidenciada nenhuma influência sobre o endotélio corneano. O autor conclui que a Metilcelulose a 2 por cento pode causar alterações tipo inflamatórias de curta duração na câmara anterior do olho de coelho, sem deixar sequela ou dano aparente às estruturas intra-oculares.

Contagem eletrônica de células endoteliais da córnea: uma adaptaçäo nacional simplificada e de baixo custo / Electronic count of corneal endothelial cells: a simplified national adaptation and of a low cost

Baseando-se em descriçäo original anterior foi feita uma adaptaçäo destinada a facilitar a contagem de células endoteliais da córnea em preparaçäo plana, utilizando materiais mais baratos e facilmente disponíveis em nosso país. Foi usada basicamente uma máquina calculadora aritmética simples, um micro-interruptor e uma caneta "hidrocor". O aparelho pode ser utilizado também em qualquer outro procedimento que necessite de contagem numérica com controle visual