Characterization of cryopreserved primary human corneal endothelial cells cultured in human serum-supplemented media / Caracterização de células endoteliais corneanas humanas primárias criopreservadas cultivadas em meio suplementado com soro humano

ABSTRACT Purpose: To compare cryopreserved human corneal endothelial cells (HCECs) grown in human serum-supplemented media (HS-SM) with cryopreserved HCECs grown in fetal bovine serum-supplemented media (FBS-SM). Methods: Three pairs of human corneas from donors aged 8, 28, and 31 years were obtained from the eye bank. From each pair, one cornea was used to start a HCEC culture using HS-SM; the other cornea was grown in FBS-SM. On reaching confluence, the six cell populations were frozen using 10% dimethyl sulfoxidecontaining medium. Thawed cells grown in HS-SM were compared with those grown in FBS-SM with respect to morphology, growth curves, immunohistochemistry, real time-reverse transcriptase polymerase chain reaction (RT-PCR) for endothelial cell markers, and detachment time. Results: No difference in morphology was observed for cells grown in the two media before or after cryopreservation. By growth curves, cell counts after thawing were similar in both media, with a slight trend toward higher cell counts in FBS-SM. Cells grown in both the media demonstrated a similar expression of endothelial cell markers when assessed by immunohistochemistry, although HCEC marker gene expression was higher in cells grown in HS-SM than in those grown in FBS-SM as assessed by RT-PCR. With FBS-SM, there was a tendency of longer detachment time and lower cell passages. Conclusions: HS-SM was similar to FBS-SM for cryopreservation of cultured HCECs as assessed by analysis of cell morphology, proliferation, and protein expression, although marker gene expression was higher in cells grown in HS-SM than in those grown in FBS-SM. Detachment time was longer with FBS-SM and in lower passages.

RESUMO Objetivo: Comparar células endoteliais de córnea humana (HCECs) criopreservadas e cultivadas em meio suplementado com soro humano (HS-SM) com HCEC criopreservadas e cultivadas em meio suplementado com soro bovino fetal (FBS-SM). Métodos: Três pares de córneas humanas de doadores com 8, 28 e 31 anos de idade foram obtidos do banco de olhos e, de cada par, uma córnea foi utilizado para iniciar uma cultura com HS-SM e outra com FBS-SM. Ao atingir a confluência, as populações de células foram congeladas utilizando-se dimetil-sulfóxido 10% no respectivo meio de cultura. Após descongeladas, as células cultivadas em HS-SM foram comparados com as cultivadas em FBS-SM por meio de morfologia, curva de crescimento, imuno-histoquímica, reação em cadeia de Reação em cadeia da polimerase da transcrição reversa em tempo real (RT-PCR) para marcadores de células endoteliais e tempo de descolamento. Resultado: Não foram observadas diferenças morfológicas antes ou após a criopreservação. Curva de crescimento mostrou contagens celulares semelhantes em ambos os meios, com discreta tendência para um maior número em FBS-SM. As células cultivadas em ambos os meios mostraram expressão semelhante de marcadores celulares endoteliais quando avaliadas por imuno-histoquímica, embora a expressão genética de marcadores para HCEC tenha sido maior em HS-SM quando avaliado por RT-PCR. Houve uma tendência de maior tempo de descolamento com FBS-SM e passagens iniciais. Conclusões: HS-SM foi semelhante ao FBS-SM na criopreservação de HCEC cultivadas in vitro quando avaliadas por morfologia celular, proliferação celular e expressão proteica, embora a expressão genética de marcadores endoteliais tenha sido maior em células cultivadas em HS-SM quando comparadas a células cultivadas em FBS-SM. O tempo de descolamento foi maior quando utilizado FBS-SM e em passagens iniciais.

Mini-flared Kelman tip, reverse tip, and sidewinder tip with torsional phaco: a prospective randomized comparative study / Faco torsional utilizando as ponteiras Kelman mini-flared, reversa e sidewinder: um estudo prospectivo comparativo randomizado

Purpose: To compare the efficiency of surgical procedures using three phaco tip designs in torsional phacoemulsification using the bevel-down technique. Methods: In this prospective, comparative, masked study, patients were randomly assigned to have torsional coaxial microincision cataract surgery using the mini-flared 45-degree Kelman tip, reversed mini-flared 30-degree Kelman tip, or Sidewinder 30-degree Kelman tip. Clinical measurements included preoperative and 3-month postoperative corrected distance visual acuity (CDVA), endothelial cell counts (ECC), and preoperative and 1-day postoperative central corneal thickness (CCT). Intraoperative measurements included phaco time, torsional time, aspiration time, case time, cumulative dissipated energy (CDE), and balanced salt solution volume (BSS). Results: The study evaluated 150 eyes of 150 patients. Intraoperatively, there was no statistically significant difference in cumulative dissipated energy, case time, torsional time, and aspiration time between the three tip configurations. However, less phaco time was used with the mini-flared 45-degree Kelman tip (p=0.02) than that with the Sidewinder 30-degree Kelman tip or reversed mini-flared 30-degree Kelman tip. The mini-flared 45-degree Kelman tip and the reversed mini-flared 30-degree Kelman tip required significantly less balanced salt solution volume than that required by the Sidewinder 30-degree Kelman tip (p=0.009). There was no statistically significant difference in corrected distance visual acuity and endothelial cell counts between tips 3 months postoperatively (p>0.05). Conclusion: All three tips were effective with no intraoperative complications. When using torsional phacoemulsification through microincisions and the prefracture technique with the bevel-down technique, the mini-flared 45-degree Kelman tip required a lower mean phaco time than the reversed mini-flared 30-degree Kelman tip and the Sidewinder 30-degree Kelman tip. .

Objetivo: Comparar a eficácia cirúrgica da facoemulsificação com tecnologia torcional utilizando 3 modelos diferentes de ponteiras. Métodos: Neste estudo prospectivo, randomizado, mascarado, os pacientes foram aleatoriamente distribuídos para serem submetidos a cirurgia de facoemulsificação coaxial torcional utilizando a ponteira Kelman mini-flared de 45 graus, ou Kelman reversed mini-flared de 30 graus ou Kelman Sidewinder de 30 graus. Os parâmetros avaliados incluíram: acuidade visual com correção (AVCC) para longe; contagem de células endoteliais (CCE) pré-operatória e pós-operatória, ao final de 3 meses; espessura corneana central (ECC) pré-operatória e no primeiro dia pós-operatório. Medidas intraoperatórias incluíram tempo de facoemulsificação, tempo de energia torcional, tempo da aspiração, tempo cirúrgico, energia dissipada acumulada (CDE) e volume de solução salina balanceada (BSS). Resultados: Este estudo avaliou 150 olhos de 150 pacientes. No intraoperatório, não foram observadas diferenças significativas na energia dissipada acumulada, tempo de facoemulsificação, tempo de energia torcional, e tempo de aspiração entre os 3 modelos de ponteira. No entanto, foi utilizando menos tempo de faco com a ponteira Kelman mini-flared de 45 graus (p=0,02) quando comparado às ponteiras Kelman Sidewinder de 30 graus e reversa mini-flared de 30 graus. A ponteira Kelman mini-flared de 45 graus e a reversa mini-flared de 30 graus utilizaram menos solução salina balanceada quando comparado à ponteira Sidewinder de 30 graus (p=0,009). Não foram observadas diferenças significativas na acuidade visual com correção, contagem de células endoteliais e espessura corneana central entre as diferentes ponteiras ao final do estudo (p=0,05). Conclusão: As 3 ponteiras foram eficazes e não apresentaram complicacões intraoperatórias. Quando foi utilizando o faco torcional através de microincisão com a técnica da pré-fratura, a ponteira Kelman mini-flared ...

Técnica de separação da membrana de Descemet para transplante de células endoteliais da córnea: estudo experimental em coelhos / Technique for separating Descemet membrane for corneal endothelial cells transplantation: experimental study in rabbits

OBJETIVO: Avaliar a porcentagem de dano endotelial induzido por uma técnica cirúrgica para a separação da membrana de Descemet contendo endotélio sadio, analisar a viabilidade e eficácia desta técnica, e avaliar a porcentagem de dano endotelial causado pela inversão da córnea em câmara anterior artificial. MÉTODOS: As córneas de três grupos de 12 coelhos da linhagem Nova Zelândia foram avaliadas. O grupo 1 foi usado como controle; portanto, as córneas foram analisadas após coletadas e trepanadas. O grupo 2 foi analisado após a inversão da córnea (endotélio para cima na posição convexa), montada em câmara anterior artificial, para o cálculo da porcentagem do dano endotelial induzido por esta inversão. O grupo 3 foi avaliado após a separação entre a membrana de Descemet e o estroma com o uso de substância viscoelástica em córneas invertidas e montadas em câmara anterior artificial. O dano endotelial foi avaliado por meio de fotografias digitais tiradas no microscópio após impregnar o endotélio com vermelho de alizarina. Amostras do grupo 3 foram processadas para avaliação histopatológica. RESULTADOS: O grupo 3 (separação viscoelástica) apresentou um índice de lesão celular endotelial de 10,06 por cento, o grupo 2 apresentou um índice de 3,58 por cento e o grupo controle um índice de 0,18 por cento de lesão celular endotelial (p<0,05). A avaliação histológica das córneas do grupo 3 revelou que aproximadamente 120 µm de espessura estromal manteve-se aderido à membrana de Descemet. CONCLUSÃO: Esta técnica deve ser melhor investigada, pois é uma alternativa viável e eficaz de separação da membrana de Descemet para transplante celular endotelial, já que o porcentual de dano celular induzido é de 10,06 por cento. A porcentagem de dano celular causado pela inversão da córnea em câmara anterior artificial foi de 3,58 por cento.

PURPOSE: To evaluate the percentage of endothelial cell damage induced during a surgical technique of Descemet's membrane separation containing healthy endothelium, analyze the viability and efficacy of this technique, and evaluate the percentage of endothelial cell damage caused by inversion of the cornea on an artificial anterior chamber. METHODS: The corneas from three groups of 12 New Zealand rabbits were evaluated. The Group one was used as the control, so the corneas were analyzed after collected and trephinated. The Group two was analyzed after inversion of the cornea (endothelial side up at a convex shape) mounted on an artificial anterior chamber to calculate the percentage of endothelial cell damage caused by this inversion. The Group three was evaluated after the separation between the Descemet's membrane and the stroma using viscoelastic substance in corneas inverted and mounted on an artificial anterior chamber. The endothelial cell damage was analyzed by digital photographs taken under a microscope after staining the endothelium with alizarin red. Group three samples were processed for histologic evaluation. RESULTS: The Group three (viscoelastic separation) showed an index of endothelial cell damage of 10.06 percent, the Group two showed an index of 3.58 percent and the control group an index of 0.18 percent of endothelial cell damage (p<0.05). Histological evaluation of the Group three corneas revealed that approximately a 120 µm thickness of stroma remained attached to the Descemet's membrane. CONCLUSION: This technique should be better investigated because it is a viable and efficient alternative of Descemet's membrane separation for endothelial cells transplantation, since the percentage of induced cell damage is 10.06 percent. The percentage of endothelial cell damage caused by inversion of the cornea on an artificial anterior chamber was 3.58 percent.

Characterization of Immortalized Human Corneal Endothelial Cell Line using HPV 16 E6/E7 on Lyophilized Human Amniotic Membrane

PURPOSE: To establish the immortalized human corneal endothelial cell line (IHCEn) by transducing human papilloma virus (HPV) 16 E6/E7 oncogenes, and to identify their characteristics when cultivated on a lyophilized human amniotic membrane (LAM). METHODS: Primary human corneal endothelial cells (PHCEn) were infected using a retroviral vector with HPV 16 E6/E7, and transformed cells were clonally selected by G418. Growth properties and characteristics of IHCEn were compared with PHCEn by cell counting and RT-PCR of VDAC3, SLC4A4, CLCN3, FGF-1, Col IV, and Na+/K+ ATPase. IHCEn were cultured on LAM. Messenger RNA expressions of VDAC3, CLCN3, and Na+/K+ ATPase, and protein expressions of Na+/K+ ATPase and Col IV in IHCEn cultivated on LAM were investigated by RT-PCR, immunofluorescence, and immunohistochemical staining, respectively. RESULTS: Successful immortalization was confirmed by stable expression of HPV 16 E6/E7 mRNA by RT-PCR, and IHCEn exhibited typical corneal endothelial morphology. Doubling time of IHCEn was 30.15+/-10.96 hrs. Both IHCEn and PHCEn expressed VDAC3, CLCN3, SLC4A4, FGF-1, Col IV, and Na+/K+ ATPase. IHCEn cultivated on LAM showed stronger expression of VDAC3, CLCN4, and Na+/K+ ATPase mRNA than on plastic culture dish. Immunohistochemical staining and immunofluorescence revealed the positive expression of Na+/K+ ATPase and Col IV. CONCLUSIONS: IHCEn were successfully established, and LAM is a good substrate for the culture of human corneal endothelial cells.

Nerve growth factor modulate proliferation of cultured rabbit corneal endothelial cells and epithelial cells / 华中科技大学学报（医学）（英德文版）

In order to investigate the effect of nerve growth factor (NGF) on the proliferation of rabbit corneal endothelial cells and epithelial cells, the in vitro cultured rabbit corneal endothelial cells and epithelial cells were treated with different concentrations of NGF. MTT assay was used to examine the clonal growth and proliferation of the cells by determining the absorbency values at 570 nm. The results showed that NGF with three concentrations ranging from 5 U/mL to 500 U/mL enhanced the proliferation of rabbit corneal endothelial cells in a concentration-dependent manner. 50 U/mL and 500 U/mL NGF got more increase of proliferation than that of 5 U/mL NGF did. Meanwhile, 50 U/mL and 500 U/mL NGF could promote the proliferation of the rabbit corneal epithelial cells significantly in a concentration-dependent manner. However, 5 U/mL NGF did not enhance the proliferation of epithelial cells. It was suggested that exogenous NGF can stimulate the proliferation of both rabbit corneal endothelial and epithelial cells, but the extent of modulation is different.

Effects of Hyaluronic Acid on the Polymorphonuclear Leukocyte (PMN) Release of Active Oxygen and Protection of Bovine Corneal Endothelial Cells from Activated PMNs

The goal of this study was to evaluate the function of hyaluronic acid (HA) on the active oxygen release from polymorphonuclear leukocytes (PMNs) and the protective effect of bovine corneal endothelial cells (BCEC) from activated PMNs. We used HA with three different molecular weights (MW 700, 000, 2, 000, 000, and 4, 000, 000) and five different concentrations (0, 0.1, 1, 2, and 3 mg/ml). We evaluated the amount of released superoxide from activated PMNs by using dismutase-inhibitable ferricytochrome C reduction. To compare the property and protective effect of HA with those of other viscoelastic substances, we used the same concentration of methylcellulose. HA suppressed superoxide release from PMNs and protected BCEC from activated PMNs in a dose-dependent, rather than a molecular weight-dependent, manner. The effect of HA reached almost a plateau at concentration above 2 mg/ml. However, methylcellulose, another viscoelastic substance, showed a similar effect. Therefore, it seems that the suppression of superoxide released from PMNs is not a property that is unique to HA, but is a general property of viscoelastic substances. Our results indicate that the action mechanism of HA proceeds not only through cell surface HA-receptor. We think that HA also acts as a physical barrier and/or a scavenger of superoxide.

The mucinous layer of corneal endothelial cells

PURPOSE: The goal of this study was to characterize the morphology of the mucinous layer on rabbit, bovine, owl, and human corneal endothelial cells. METHODS: Corneoscleral buttons were fixed using cetylpyridinium chloride to stabilize "mucus" and the tissue was prepared for transmission electron microscopy. Photomicrographs were measured to determine the thickness of the endothelial and epithelial mucinous layer in the central cornea. RESULTS: The endothelial mucinous layer was seen as a nearly uniform electrodense region on the apical aspect of the endothelium. It was found to be 0.9 microm, 0.9 microm, 0.9 microm, and 0.5 microm thick in rabbit, bovine, owl, and human, respectively. The owl endothelium had an additional less electrodense layer with a granular appearance and a thickness of about 200 microm. The mucinous layer on the epithelium was similar in appearance to that on the endothelium and across species. CONCLUSIONS: The morphologic similarity of the endothelial and epithelial mucinous layers is a serendipitous finding that should prove valuable in experimental design. Ultimately, it is hoped that studies of the posterior corneal surface will deepen our knowledge of endothelial protection.

Açäo da poliacrilamida a 4,5 por cento na câmara anterior de olhos de coelhos: estudo da paquimetria, da contagem das células endoteliais, da tonometria, da biomicroscopia e da histologia / The effect of 4,5 per cent polyacrylamide in the anterior chamber of the rabbits: study of pachometry, endothelial cell count, tonometry, biomicroscopy and histology

Verificou-se a açäo da poliacrilamida a 4,5 por cento na câmara anterior de 46 coelhos, divididos em 3 grupos: grupo 1- composto de 20 animais onde se avaliou a paquimetria ultrasonica e contagem das células do endotélio corneano; grupo 2- composto de 20 animais onde se avaliou a tonometria de aplanaçäo e a biomicroscopia da câmara anterior e grupo 3- composto de 6 animais onde se avaliou a histologia de cortes realizados em vários planos. O humor aquoso do olho direito foi substituído parcialmente (0,12ml) por poliacrilamida a 4,5 por cento e o do olho esquerdo, no mesmo volume, por soluçäo salina balanceada (olho-controle). A poliacrilamida produziu aumento da espessura corneana semelhante à soluçäo salina balanceada e ambas näo tiveram influência sobre a densidade das células endoteliais. A poliacrilamida está relacionada com hipertensäo ocular nas primeiras 24 horas, com pico entre a 2ª e a 4ª hora de observaçäo. A biomicroscopia e a histologia mostraram que a poliacrilamida está relacionada com maior número de células, maior concentraçäo de proteinas (flare) no humor aquoso e maior hiperemia iriana, em maior número de olhos, com maior intensidade e por tempo mais prolongado que a soluçäo salina balanceada. A poliacrilamida produziu grumos na câmara anterior e sobre a íris além de precipitados retroceráticos e sobre a cápsula anterior do cristalino, provavelmente relacionados com o acúmulo e a condensaçäo da droga. O autor concluiu que a poliacrilamida a 4,5 por cento pode causar aumento da espessura corneana por distúrbio funcional reversível do endotélio corneano, hipertensäo ocular e reaçäo inflamatória na câmara anterior de curta duraçäo, em olhos de coelho, sem deixar sequela ou dano aparente às estruturas intra-oculares.

Heterologous corneal endothelial cell transplantation: human corneal endothelial cell transplantation in Lewis rats

A heterologous corneal endothelial transplantation was attempted using human endothelial cells and a Lewis rat penetrating keratoplasty model. Cultured human endothelial cells were seeded to a Lewis rat cornea, which was denuded of its endothelium. When grafted into the syngeneic Lewis rat, the graft remained clear for at least five days, and then became opaque and edematous because of immune rejection reaction. In contrast, corneas denuded of their endothelium became opaque and edematous immediately after transplantation. These results demonstrate that transplanted endothelial cells have enough antigens to induce rejection reaction even though they have the functional capacity to deturge the cornea.

Estudo experimental do meio de conservaçäo de córneas de McCarey-Kaufman modificado / Experimental study of the media for corneal preservation of modified McCarey-Kaufman

Este trabalho relata o estudo experimental do meio de conservaçäo de córneas de McCarey-Kaufman modificado produzido no Hospital das Clínicas da Faculdade de Medicina da Universidade de Säo Paulo (meio "MKM-HC") na conservaçäo de córneas de coelhos e humanas, tendo-se demonstrado a eficácia deste meio na conservaçäo destas córneas para transplantes por um período de até 96 horas

Clinical experience of hydrogel soft intraocular lenses

Twenty-six soft intraocular lenses were implanted in twenty-six senile cataract patients from luly 16, 1987, to April 15, 1988 at Kangnam St. Mary's Hospital, Seoul, Korea. The average age of the patients was seventy-six with a range from fifty-five to eight nine years old. Of the twenty-six patients at the 12 months follow up 87.5% have arhieved visual acuity of 20/40 or better. The average central corneal endothelial cell loss at postoperative 7 days and 3 months were 9.8% and 12.3% respectively. Complications were observed in six patients. Two patients had preexisting retinal and optic diseases and one had a fibrinous membrane. Three patients had transient pigmentary precipitates on IOL surface.

A prospective clinical study of radial keratotomy in Koreans

Three hundred and forty-eight eyes (246 patients, both eye radial keratototmy in 102 patients) which could be followed-up for at least one year or more were included in this study. Postoperative uncorrected visul acuity which was 20/40 or more could be obtained in 79% of the lower myopic eyes (-1.75--2.75 D), in 73% of the moderate myopic eyes (-3.00--5.75 D) and in 34% of high myopic eyes (-6.00 D-). The postoperative refractive correction (spherical equivalent) ranged from plano to -9.0 D with a mean decrease of -3.86 D with a mean reduction of keratometry in moderate myopia of 3.33 D with a range from 0.5 D to 6.75 D. But its mean reduction in high myopia did not parallel its myopic degree. Glare and fluctuation of vision were the most frequent complaints following surgery. Some patients had continuous constant vision improvement for a long period while some patients (or other eyes) had episodes of decreased vision because of the recurrence of myopia or hyperopia shift. The mean central corneal endothelial cell loss determined 6-l2 months later was 5.31-5.61%. Microperforation in 11 eyes (3%) occurred during the early part of the study and improved naturally without any problem. An over-correction of more than +1.0 D (2%) and an under-correction of more than -1.0 D (1%) with having induced or residual astigmatism (17%) were observed. Radial keratotomy is suceessful in carefully selected patients with mild and moderate myopia, and also in anisometropic high myopia.

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.