A Framework for Human Corneal Endothelial Cell Culture and Preliminary Wound Model Experiments with a New Cell Tracking Approach.

Cell injection therapy is emerging as an alternative to treat corneal endothelial dysfunction (CED) and to avoid corneal scarring due to bullous keratopathy. However, establishing a standardized culture procedure that provides appropriate cell yield while retaining functional features remains a challenge. Here, we describe a detailed framework obtained from in vitro culture of human corneal endothelial cells (HCECs) and comparative in vivo experimental models for CED treatment with a new cell tracking approach. Two digestion methods were compared regarding HCEC morphology and adhesion. The effect of Y-27632 (ROCKi) supplementation on final cell yield was also assessed. Cell adhesion efficacy with two cell delivery systems (superparamagnetic embedding and cell suspension) was evaluated in an ex vivo human cornea model and in an in vivo rabbit CED model. The injection of supplemented culture medium or balanced salt solution (BSS) was used for the positive and negative controls, respectively. HCEC isolation with collagenase resulted in better morphology and adhesion of cultured HCEC when compared to EDTA. Y-27632 supplementation resulted in a 2.6-fold increase in final cell yield compared to the control. Ex vivo and in vivo adhesion with both cell delivery systems was confirmed by cell tracker fluorescence detection. Corneal edema and opacity improved in both animal groups treated with cultured HCEC. The corneas in the control groups remained opaque. Both HCEC delivery systems seemed comparable as treatments for CED and for the prevention of corneal scarring.

Analysis of different conditioned media secreted by limbal progenitor cells in the modulation of corneal healing.

Ex vivo cultivation and transplantation of limbal epithelial cells has been reported as an alternative source for ocular surface reconstruction. However, until now, the functional improvement of these patients is limited due to the low survival rate of the transplanted cells. Consequently, the clinical benefits of this therapeutic strategy are only temporary and can assign them to paracrine effects associated with the transplanted cells. With this background in mind, we aimed to analyze the effect of different conditioned media containing growth factors secreted by limbal progenitor cells on corneal epithelial healing, both in vitro and in vivo. Limbal tissue was used to obtain different conditioned media (CM). For the in vitro assay, corneal epithelial cells were treated with CM and the epithelial migration was analyzed. Growth factors in the CM were identified with ELISA and multiplex. For the in vivo assay in rats, total limbal stem cell deficiency (LSCD) was induced with an abrasive injury to the ocular surface, and the animals were treated with different CM. Clinical and histological analyses were performed. In the in vitro assay, treatment with limbal fibroblast (LF CM) was more effective compared to the other CM, and analysis revealed high concentrations of keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF). In the in vivo assay, animals treated with LF CM showed epithelial defect improvement, maintenance of thickness, and decreased opacity and neovascularization. This treatment also allowed better ocular surface tissue organization when compared to the other treatments. The in vitro and in vivo experiments showed better outcomes in the corneal wound healing for the LF CM treatment. The high concentrations of KGF and HGF, linked to epithelial cell migration and proliferation, may correlate to the best results found in this treatment.

Mass variability in drops of multidose eyedrops: a quality and reliability study / Variabilidade da massa das gotas dos colírios: um estudo de qualidade e confiabilidade

ABSTRACT Purpose: This quality and reliability study aimed to identify the mass variability of multidose eyedrops and to verify the existence of a reference pattern for the drop volume of eyedrops using standard lubricant eyedrops available on the Brazilian market. Methods: Five brands of lubricant eyedrops were evaluated. An ideal standard 20 µL drop of eyedrops from each manufacturer was captured using an adjustable micropipette. The eyedrop bottles were randomly selected, and five measurements of the samples' masses were collected using calibrated precision scales. Results: The mass of the 20 µL samples varied significantly (p<0.001) among the different manufacturers. However, among eyedrops of the same brand, mass variation was not statistically different. The global mean mass of all weighed drops was 18.24 mg, and non-uniformity was identified across all eyedrop brands. Conclusion: Significant variations in the drop masses of common lubricant eyedrops were identified using standard laboratory equipment. Heterogeneity in the drop volume of standard eyedrop medications suggest that potential dosage discrepancies exist, possibly altering treatment efficacy. A pre-established reference measure may lead to the production of more appropriately sized eyedrops for use in human eyes.

RESUMO Objetivo: Este estudo de qualidade e confiabilidade teve como objetivo identificar a variabilidade da massa do volume da gota de colírios multidose e verificar a existência de um padrão de referência para o volume da gota dos colírios usando lágrimas artificiais do mercado brasileiro. Métodos: Cinco marcas de colírios lubrificantes foram avaliadas quanto ao volume da gota. Uma gota padrão ideal de 20 µL de cada fabricante foi coletada usando uma micropipeta ajustável. Os frascos dos colírios foram selecionados aleatoriamente e cinco medidas das massas das amostras foram coletadas usando escalas de precisão calibradas. Resultados: A massa das amostras de 20 µL variou significativamente (p<0,001) entre os diferentes fabricantes. No entanto, entre os colírios da mesma marca, a variação da massa não foi estatisticamente diferente. A massa média global de todas as gotas pesadas foi de 18,24 mg e foi observada uma não-uniformidade entre todas as marcas de colírios. Conclusão: Identificou-se uma variação significativa nas massas do volume das gotas dos colírios lubrificantes, usando equipamento padrão de laboratório. A heterogeneidade no volume da gota dos colírios testados sugere a existência de discrepâncias potenciais em suas posologias, possivelmente alterando a eficácia do tratamento. Uma medida de referência pré-estabelecida pode levar à produção de colírios com gotas de tamanho mais apropriado para uso em olhos humanos.

Mass variability in drops of multidose eyedrops: a quality and reliability study.

PURPOSE: This quality and reliability study aimed to identify the mass variability of multidose eyedrops and to verify the existence of a reference pattern for the drop volume of eyedrops using standard lubricant eyedrops available on the Brazilian market. METHODS: Five brands of lubricant eyedrops were evaluated. An ideal standard 20 µL drop of eyedrops from each manufacturer was captured using an adjustable micropipette. The eyedrop bottles were randomly selected, and five measurements of the samples' masses were collected using calibrated precision scales. RESULTS: The mass of the 20 µL samples varied significantly (p<0.001) among the different manufacturers. However, among eyedrops of the same brand, mass variation was not statistically different. The global mean mass of all weighed drops was 18.24 mg, and non-uniformity was identified across all eyedrop brands. CONCLUSION: Significant variations in the drop masses of common lubricant eyedrops were identified using standard laboratory equipment. Heterogeneity in the drop volume of standard eyedrop medications suggest that potential dosage discrepancies exist, possibly altering treatment efficacy. A pre-established reference measure may lead to the production of more appropriately sized eyedrops for use in human eyes.

Microbial Cross-contamination in Multidose Eyedrops: The Impact of Instillation Angle and Bottle Geometry.

Purpose: To evaluate the impact of instillation angle and nozzle tip geometry on cross-contamination risk of multidose ocular solution bottles. Methods: Pseudomonas aeruginosa solution was passed exclusively on the outside of the nozzle to simulate contamination on the exterior of topical agents. Three drops were administered from angles of 90° and 45° from bottles with either a round or sharp tip geometry, and the cultures were examined for growth. Two-hundred sixteen cultures from nine lubricant eyedrop brands currently existing in the Brazilian market were assessed for bacterial growth. Results: After seven days, bacterial contamination was detected in 53.7% of cultures when drops were administered at 90° and in 70.4% of cultures at 45°. Eyedrops collected from a rounded nozzle tip and an instillation angle of 90° transmitted bacteria in 69.4% of cases, whereas those administered from a sharp tip transmitted bacteria in only 22.2% of cases (P = 0.001). At an instillation angle of 45°, contamination was identified in 83.3% of bottles with a rounded tip geometry and in only eight of 18 bottles (44.4%) from those with a sharp nozzle geometry (P = 0.005). Conclusions: Adjusting the instillation angle of eyedrop solutions to 90°, as well as using a nozzle geometry that prevents flow of the solution to the side of the bottle, significantly reduced contamination rates. Translational Relevance: Standardizing drop bottles and adjusting delivery angle shows promise in reducing contamination rates and may critically impact the quality of care for patients requiring topical therapeutic agents.

Piezoelectric 3D bioprinting for ophthalmological applications: process development and viability analysis of the technology.

Piezoelectric inkjet 3D bioprinting technology is a viable technique for ophthalmological applications. It provides versatility, high sensibility and accuracy, required in ophthalmological procedures. A process flow for biofabrication was described in detail and validated, using piezoelectric inkjet technology, for ophthalmological applications, in vitro and in situ, based on complex images. Ophthalmological problems were documented by diagnostic examinations and were fed to the flow as complex images. The Concept Mapping methodology and the Conceptual Design approach were utilized to elaborate the 3D bioprinting process flow. It was developed a bioink with corneal epithelial cells. To simulate an in situ bioprinting process, eyes of pigs were selected as the substrate to print the cells. Print scripts used the digitally treated images. In order to print on predefined locations, alignment devices and sample holders were built. The proposed process flow has shown to be a potential tool for the biofabrication of ophthalmological solutions.

Amniotic membrane as a biological scaffold for dental pulp stem cell transplantation in ocular surface reconstruction / Membrana amniótica como substrato biológico para o transplante de células-tronco da polpa do dente de leite na reconstrução da superfície ocular

ABSTRACT Purpose: To evaluate the ability of human immature dental pulp stem cells, which are mesenchymal stem cells of neural crest origin, to differentiate into the corneal epithelium for purposes of corneal transplantation and tissue engineering when cultured on de-epithelized amniotic membranes. Methods: We compared the immunophenotypes (ABCG2, K3/12, and vimentin) of cells grown on amniotic membranes or plastic surfaces under serum-free conditions or in culture media containing serum or serum replacement components. Results: Immature dental pulp stem cells grown on amniotic membranes under basal conditions are able to maintain their undifferentiated state. Our data also suggest that the culture medium used in the present work can modulate the expression of immature dental pulp stem cell markers, thus inducing epithelial differentiation of these cells in vitro. Conclusions: Our results suggest that the amniotic membrane is a good choice for the growth and transplantation of mesenchymal stem cells, particularly immature dental pulp stem cells, in clinical ocular surface reconstruction.

RESUMO Objetivos: Avaliar a capacidade das células-tronco imaturas da polpa do dente de leite que são células-tronco mesenquimais de origem da crista neural, de se diferenciarem no epitélio corneano para fins de transplante de córnea e engenharia de tecidos quando cultivadas em membrana amnióticas desepitelizadas. Métodos: Foram comparamos so imunofenótipo (ABCG2, CK3/12 e vimentina) de células cultivadas em membranas amnióticas ou em superfícies plásticas sob condições livres de soro ou em meios de cultura contendo soro ou componentes de substituição de soro. Resultados: Células-tronco imaturas da polpa do dente de leite cultivadas sobre membrana amniótica em condições basais são capazes de manter seu estado indiferenciado. Nossos dados também sugerem que o meio de cultura utilizado no presente trabalho pode modular a expressão de marcadores de células-tronco imaturas da polpa do dente de leite, induzindo a diferenciação epitelial destas células in vitro. Conclusão: Nossos resultados sugerem que a membrana amniótica é uma boa escolha para o crescimento e transplante de células-tronco mesenquimais, particularmente as células-tronco imaturas da polpa do dente de leite, na reconstrução da superfície ocular.

Evaluating the diameter of eyedropper tips using a computer vision system / Avaliação do diâmetro de bicos de conta-gotas de colírios por visão computacional

ABSTRACT Purpose: This study aimed to determine the variation in diameters of outer and inner apertures of eyedropper tips using a computer vision system. Standardizing the size of eye drop nozzles is crucial to reduce the treatment cost of chronic eye diseases and to ensure a continued use of medication. An eyedropper volume of >20 µL (maximum storage of the conjunctival sac) causes medication wastage and increases treatment costs. Methods: We measured the diameters of the outer and inner apertures of eyedropper tips and evaluated variations in diameters using a computerized visual inspection system. Results: The computer visual inspection system identified anomalies in the apertures of eyedropper tips that resulted in diameter variations. Conclusions: The results of the present study show discrepancies in diameters of eyedropper tips, suggesting a variation in eyedropper size and medication wastage.

RESUMO Objetivo: Este estudo teve como objetivo determinar a variação dos diâmetros das aberturas externa e interna dos bicos conta-gotas utilizando sistema de visão computacional. A padronização do tamanho dos colírios conta-gotas é importante para reduzir o custo do tratamento de doenças crônicas e garantir o uso contínuo de medicamentos. O volume da gota maior do que 20 µl (volume de armazenamento máximo do saco conjuntival) gera desperdício da medicação e aumenta o custo do tratamento. Métodos: Medimos os diâmetros das aberturas externa e interna das pontas dos conta-gotas e avaliamos as variações no diâmetro usando um sistema de inspeção visual computadorizado. Resultados: O sistema de inspeção visual por computador identificou anomalias nas aberturas dos bicos dos frascos conta-gotas que resultaram em variações de diâmetro. Conclusões: Os resultados do presente estudo mostram discrepâncias nos diâmetros dos bicos dos frascos dos conta-gotas, sugerindo uma variação no tamanho das gotas e no desperdício de remédios.

Evaluating the diameter of eyedropper tips using a computer vision system.

PURPOSE: This study aimed to determine the variation in diameters of outer and inner apertures of eyedropper tips using a computer vision system. Standardizing the size of eye drop nozzles is crucial to reduce the treatment cost of chronic eye diseases and to ensure a continued use of medication. An eyedropper volume of >20 µL (maximum storage of the conjunctival sac) causes medication wastage and increases treatment costs. METHODS: We measured the diameters of the outer and inner apertures of eyedropper tips and evaluated variations in diameters using a computerized visual inspection system. RESULTS: The computer visual inspection system identified anomalies in the apertures of eyedropper tips that resulted in diameter variations. CONCLUSIONS: The results of the present study show discrepancies in diameters of eyedropper tips, suggesting a variation in eyedropper size and medication wastage.

Amniotic membrane as a biological scaffold for dental pulp stem cell transplantation in ocular surface reconstruction.

PURPOSE: To evaluate the ability of human immature dental pulp stem cells, which are mesenchymal stem cells of neural crest origin, to differentiate into the corneal epithelium for purposes of corneal transplantation and tissue engineering when cultured on de-epithelized amniotic membranes. METHODS: We compared the immunophenotypes (ABCG2, K3/12, and vimentin) of cells grown on amniotic membranes or plastic surfaces under serum-free conditions or in culture media containing serum or serum replacement components. RESULTS: Immature dental pulp stem cells grown on amniotic membranes under basal conditions are able to maintain their undifferentiated state. Our data also suggest that the culture medium used in the present work can modulate the expression of immature dental pulp stem cell markers, thus inducing epithelial differentiation of these cells in vitro. CONCLUSIONS: Our results suggest that the amniotic membrane is a good choice for the growth and transplantation of mesenchymal stem cells, particularly immature dental pulp stem cells, in clinical ocular surface reconstruction.

Amniotic membrane as a biological scaffold for dental pulp stem cell transplantation in ocular surface reconstruction

Purpose: To evaluate the ability of human immature dental pulp stem cells, which are mesenchymal stem cells of neural crest origin, to differentiate into the corneal epithelium for purposes of corneal transplantation and tissue engineering when cultured on de-epithelized amniotic membranes. Methods: We compared the immunophenotypes (ABCG2, K3/12, and vimentin) of cells grown on amniotic membranes or plastic surfaces under serum-free conditions or in culture media containing serum or serum replacement components. Results: Immature dental pulp stem cells grown on amniotic membranes under basal conditions are able to maintain their undifferentiated state. Our data also suggest that the culture medium used in the present work can modulate the expression of immature dental pulp stem cell markers, thus inducing epithelial differentiation of these cells in vitro. Conclusions: Our results suggest that the amniotic membrane is a good choice for the growth and transplantation of mesenchymal stem cells, particularly immature dental pulp stem cells, in clinical ocular surface reconstruction.

Amniotic membrane as a biological scaffold for dental pulp stem cell transplantation in ocular surface reconstruction

Purpose: To evaluate the ability of human immature dental pulp stem cells, which are mesenchymal stem cells of neural crest origin, to differentiate into the corneal epithelium for purposes of corneal transplantation and tissue engineering when cultured on de-epithelized amniotic membranes. Methods: We compared the immunophenotypes (ABCG2, K3/12, and vimentin) of cells grown on amniotic membranes or plastic surfaces under serum-free conditions or in culture media containing serum or serum replacement components. Results: Immature dental pulp stem cells grown on amniotic membranes under basal conditions are able to maintain their undifferentiated state. Our data also suggest that the culture medium used in the present work can modulate the expression of immature dental pulp stem cell markers, thus inducing epithelial differentiation of these cells in vitro. Conclusions: Our results suggest that the amniotic membrane is a good choice for the growth and transplantation of mesenchymal stem cells, particularly immature dental pulp stem cells, in clinical ocular surface reconstruction.

Minor Salivary Gland Transplantation for Severe Dry Eyes.

Dry eye is a multifactorial disease comprising a wide spectrum of ocular surface alterations and symptoms of discomfort. In most patients with aqueous-deficient dry eye, pharmaceutical tear substitutes are used to control symptoms and prevent ocular surface damage. However, in severe dry eye conditions caused by cicatricial disorders, such as Stevens-Johnson syndrome and ocular cicatricial mucous membrane pemphigoid, noninvasive treatments are insufficient, and patients are at risk of developing complications that can lead to blindness. The use of salivary glands as a source of lubrication to treat severe cases of dry eye has been proposed by different authors. The first reports proposed parotid or submandibular gland duct transplantation into the conjunctival fornix. However, complications limited the functional outcomes. Minor salivary gland autotransplantation together with labial mucosa has been used as a complex graft to the conjunctival fornix in severe dry eye with a good outcome. Our group demonstrated significant improvements in best-corrected visual acuity, Schirmer I test score, corneal transparency, and neovascularization after using this technique. A symptoms questionnaire applied to these patients revealed improvements in foreign body sensation, photophobia, and pain. Similar to tears, saliva has a complex final composition comprising electrolytes, immunoglobulins, proteins, enzymes, and mucins. We demonstrated the viability of minor salivary glands transplanted into the fornix of patients with dry eye by performing immunohistochemistry on graft biopsies with antibodies against lactoferrin, lysozyme, MUC1, and MUC16. The findings revealed the presence of functional salivary gland units, indicating local production of proteins, enzymes, and mucins.

Variação do volume de gotas de colírios lubrificantes disponíveis no mercado brasileiro / Variation in the volume of lubricating eyedrops available in the brazilian market

RESUMO Objetivo: Avaliar a variação intra e interexaminadores do volume de gotas dispensados de frascos de colírios lubrificantes disponíveis no mercado. Métodos: Foram estudados cinco frascos de colírios lubrificantes e dezenove voluntários participaram deste estudo. A massa média de gotas de 20µl dos colírios foi obtida utilizando micropipeta e balança de precisão e como padrão para comparação com a massa das gotas obtidas pelos voluntários. Cinco gotas de cada frasco foram pesadas individualmente com o tubo de colírio perpendicular à balança, usando o primeiro e segundo dedos da mão direita, de forma que a pressão 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 colírios apresentaram variação estatisticamente significante das massas das gotas obtidas pelos examinadores quando comparadas com a massa média padrão de 0,0182±0,0014g, com exceção da comparação entre os dados do colírio A com o colírio D, que não apresentou variação estatisticamente significante. Conclusão: O presente estudo demonstra a ausência de uniformidade das gotas dispensadas pelos frascos de colírios disponíveis no mercado e a sua inadequação à real necessidade, uma vez que as gotas dispensadas são maiores do que o indicado. Esse fato torna-se um problema quando se trata de período de tratamento prolongado, especialmente com colírios dispendiosos como os indicados para a terapêutica do glaucoma. Nesse sentido, a padronização das gotas de colírios se faz necessária.

ABSTRACT Objective: To evaluate the intra and inter variations of eye drops volume dispensed from bottles available on the market. Methods: Five bottles of lubricant eye drops were studied and nineteen volunteers participated in this study. The average mass from 20µl of eye drops was obtained using accuracy micropipette and balance, and used as standard for comparison with the mass of the drops obtained by the volunteers. Five drops of each vial were individually weighed with the tube perpendicular to the balance, using the first and second fingers of the right hand, so that the pressure was applied only in the middle of the flask. The experiments were performed in a room temperature (21±1°C). Results: All eye drops bottles showed a statistically significant variation on masses of the drops obtained by examiners when compared with the standard average weight of 0.0182±0,0014g, except when compared A with D eye drops, with no statistically significant variation. Conclusion: This study demonstrates the lack of uniformity of drops dispensed by eye drops bottles available in the market and its inadequacy to the real need, since the dispensed drops are larger than indicated.This fact becomes a problem when it comes to long treatment period, especially with expensive drops as indicated for glaucoma therapy. In this sense, the standardization of drops of eye drops is necessary.

Cytokine Dosage in Fresh and Preserved Human Amniotic Membrane.

PURPOSE: To evaluate cytokine concentrations in amniotic membrane (AM) preserved in different preservation media, temperatures, and times and to compare them with those in fresh AM. METHODS: Placentas were harvested from 8 women undergoing cesarean delivery, with each then divided into 17 pieces for the following preservation methods: at 2 different temperatures (-80 and 0°C), in 2 different preservation media (dimethyl sulfoxide and enriched TC199; Ophthalmos), and for different time periods (for 1, 7, 60, and 180 days). Nonpreserved fresh AM was used as a control. An enzyme-linked immunosorbent assay was performed on the supernatant for detection of the following cytokines: epidermal growth factor, basic fibroblast growth factor, hepatocyte growth factor, keratinocyte growth factor, transforming growth factor-beta, and interleukins 4 and 10, and the findings were assessed by post hoc analysis of variance. RESULTS: AM preserved at -80°C showed less decrease in the concentration of 4 cytokines. Three cytokines showed less decrease in AM preserved in the TC199 medium, whereas 1 showed less decrease in AM preserved in dimethyl sulfoxide. After storage, 5 cytokine concentrations remained stable for up to 1 day, 3 remained stable for up to 7 days, and all showed significant loss thereafter. CONCLUSIONS: The AM storage temperature of -80°C was found optimal for maintaining the concentrations of most of the tested cytokines, and enriched TC199 medium was the optimal long-term storage medium for maintaining the concentration of 3 of the cytokines, and with less decrease. When possible, AM should be used within 1 to 7 days after harvesting.

Genetic analysis and cAMP measurement: comparison between lean and obese anovulating mice.

PURPOSE: To evaluate genes differentially expressed in ovaries from lean (wild type) and obese (ob/ob) female mice and cyclic AMP production in both groups. METHODS: The expression on messenger RNA levels of 84 genes concerning obesity was analyzed through the PCR array, and cyclic AMP was quantified by the enzyme immunoassay method. RESULTS: The most downregulated genes in the Obesity Group included adenylate cyclase-activating polypeptide type 1, somatostatin, apolipoprotein A4, pancreatic colipase, and interleukin-1 beta. The mean decrease in expression levels of these genes was around 96, 40, 9, 4.2 and 3.6-fold, respectively. On the other hand, the most upregulated genes in the Obesity Group were receptor (calcitonin) activity-modifying protein 3, peroxisome proliferator activated receptor alpha, calcitonin receptor, and corticotropin-releasing hormone receptor 1. The increase means in the expression levels of such genes were 2.3, 2.7, 4.8 and 6.3-fold, respectively. The ovarian cyclic AMP production was significantly higher in ob/ob female mice (2,229 ± 52 fMol) compared to the Control Group (1,814 ± 45 fMol). CONCLUSIONS: Obese and anovulatory female mice have reduced reproductive hormone levels and altered ovogenesis. Several genes have their expression levels altered when leptin is absent, especially adenylate cyclase-activating polypeptide type 1.

Genetic analysis and cAMP measurement: comparison between lean and obese anovulating mice / Análise genética e dosagem de AMP cíclico: comparação entre camundongos fêmeas anovuladoras magras e obesas

PURPOSE: To evaluate genes differentially expressed in ovaries from lean (wild type) and obese (ob/ob) female mice and cyclic AMP production in both groups. METHODS: The expression on messenger RNA levels of 84 genes concerning obesity was analyzed through the PCR array, and cyclic AMP was quantified by the enzyme immunoassay method. RESULTS: The most downregulated genes in the Obesity Group included adenylate cyclase-activating polypeptide type 1, somatostatin, apolipoprotein A4, pancreatic colipase, and interleukin-1 beta. The mean decrease in expression levels of these genes was around 96, 40, 9, 4.2 and 3.6-fold, respectively. On the other hand, the most upregulated genes in the Obesity Group were receptor (calcitonin) activity-modifying protein 3, peroxisome proliferator activated receptor alpha, calcitonin receptor, and corticotropin-releasing hormone receptor 1. The increase means in the expression levels of such genes were 2.3, 2.7, 4.8 and 6.3-fold, respectively. The ovarian cyclic AMP production was significantly higher in ob/ob female mice (2,229±52 fMol) compared to the Control Group (1,814±45 fMol). CONCLUSIONS: Obese and anovulatory female mice have reduced reproductive hormone levels and altered ovogenesis. Several genes have their expression levels altered when leptin is absent, especially adenylate cyclase-activating polypeptide type 1. .

OBJETIVO: Avaliar os genes diferencialmente expressos em ovários de camundongos fêmeas magras (tipo selvagem) e obesas (ob/ob) e a produção de AMP cíclico em ambos os grupos. MÉTODOS: A expressão nos níveis de RNA mensageiro de 84 genes relacionados à obesidade foi analisada por PCR Array, e o AMP cíclico foi quantificado por método imunoenzimático. RESULTADOS: Os genes que mais sofreram diminuição da expressão no Grupo Obesidade incluíram o tipo 1 de polipeptídeo ativador da adenilato ciclase, o da somatostatina, da apolipoproteína A4, da colipase pancreática e da beta interleucina 1. A média de redução na expressão desses genes foi de aproximadamente 96, 40, 9, 4,2 e 3,6 vezes, respectivamente. Por outro lado, os genes que mais tiveram aumento na expressão no Grupo Obesidade foram o gene da proteína modificadora da atividade do receptor de calcitonina 3, do proliferador de peroxissomos ativados por proteína alfa, do receptor de calcitonina e do receptor para hormônio liberador de corticotropinas 1. As médias de acréscimo nos níveis de expressão de tais genes foram de 2,3, 2,7, 4,8 e 6,3 vezes, respectivamente. A produção de AMP cíclico ovariana foi significantemente aumentada em camundongos fêmeas ob/ob (2.229±52 fMol) quando comparada ao Grupo Controle (1.814±45 fMol). CONCLUSÕES: Camundongos fêmeas obesas e anovuladoras possuem níveis de hormônio reprodutivo reduzidos e ovulogênese alterada. Vários genes mostram níveis de expressão alterados quando a leptina está ausente, principalmente o tipo 1 de polipeptídeo ativador da adenilato ciclase. .

Comparison of culture media for ex vivo cultivation of limbal epithelial progenitor cells.

PURPOSE: To compare the effectiveness of three culture media for growth, proliferation, differentiation, and viability of ex vivo cultured limbal epithelial progenitor cells. METHODS: Limbal epithelial progenitor cell cultures were established from ten human corneal rims and grew on plastic wells in three culture media: supplemental hormonal epithelial medium (SHEM), keratinocyte serum-free medium (KSFM), and Epilife. The performance of culturing limbal epithelial progenitor cells in each medium was evaluated according to the following parameters: growth area of epithelial migration; immunocytochemistry for adenosine 5'-triphosphate-binding cassette member 2 (ABCG2), p63, Ki67, cytokeratin 3 (CK3), and vimentin (VMT) and real-time reverse transcription polymerase chain reaction (RT-PCR) for CK3, ABCG2, and p63, and cell viability using Hoechst staining. RESULTS: Limbal epithelial progenitor cells cultivated in SHEM showed a tendency to faster migration, compared to KSFM and Epilife. Immunocytochemical analysis showed that proliferated cells in the SHEM had lower expression for markers related to progenitor epithelial cells (ABCG2) and putative progenitor cells (p63), and a higher percentage of positive cells for differentiated epithelium (CK3) when compared to KSFM and Epilife. In PCR analysis, ABCG2 expression was statistically higher for Epilife compared to SHEM. Expression of p63 was statistically higher for Epilife compared to SHEM and KSFM. However, CK3 expression was statistically lower for KSFM compared to SHEM. CONCLUSIONS: Based on our findings, we concluded that cells cultured in KSFM and Epilife media presented a higher percentage of limbal epithelial progenitor cells, compared to SHEM.

Comparison between different biomaterial scaffolds for limbal-derived stem cells growth and enrichment.

PURPOSE/AIM: Corneal epithelial stem cells have been used for the treatment of total limbal deficiency with corneal conjunctivalization and decreased vision secondary to a variety of ocular surface diseases. We set to compare the ability of different extracellular components in promoting growth and migration of these cells. MATERIALS AND METHODS: Growth parameters were evaluated, including cell migration and proliferation (by wound healing) and mRNA gene expression (by quantitative RT-PCR). RESULTS: The growth of corneal epithelial cells plated onto different matrix has shown that all treatments were efficient in supporting exponential growth, with a small increase in the puramatrix and collagen I groups when compared with fibrin treatment, which displayed the best doubling time rate and saturation density. The mRNA relative levels for c-myc, a proliferation marker, were considerably higher in the fibrin-coated group. In a smaller extent, the same could be observed for the puramatrix and collagen I groups. The same pattern could be observed for ß-1 and α-6-integrin mRNA relative levels. The levels of CD71 mRNA, a LESC negative marker, were decreased in all groups, with a greater decrease in the fibrin group. We also found that the relative mRNA levels of the efflux pump ABCG2 and ΔNp63 transcripts were significantly higher in the fibrin group but not for collagen and puramatrix groups. Moreover, a diminished capacity of wound repair was observed for the uncoated control while the coated biomaterial groups were able to restore the cell-covered surface at some extent. CONCLUSION: All components tested were effective in promoting growth of corneal epithelial cells and maintenance of stem cell putative markers when compared with the uncoated surface group. Fibrin was far superior than collagen I and puramatrix in promoting survival, growth and migration of these cells.

[Comparison between amniotic membrane with and without epithelium as a support for human limbal epithelial cells cultured ex vivo]. / 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.