Fuchs endothelial corneal dystrophy: an updated review.

PURPOSE: The present review will summarize FECD-associated genes and pathophysiology, diagnosis, current  therapeutic approaches, and future treatment perspectives. METHODS: Literature review. RESULTS: Fuchs' endothelial corneal dystrophy (FECD) is the most common bilateral corneal dystrophy and accounts for one-third of all corneal transplants performed in the US. FECD is caused by a combination of genetic and non-heritable factors, and there are two types: early-onset FECD, which affects individuals from an early age and is usually more severe, and late-onset FECD, which is more common and typically manifests around the age of 40. The hallmark findings of FECD include progressive loss of corneal endothelial cells and the formation of focal excrescences (guttae) on the Descemet membrane. These pathophysiological changes result in progressive endothelial dysfunction, leading to a decrease in visual acuity and blindness in later stages. The present review will summarize FECD-associated genes and pathophysiology, diagnosis, current therapeutic approaches, and future treatment perspectives. CONCLUSION: With the characterization and understanding of FECD-related genes and ongoing research into regenerative therapies for corneal endothelium, we can hope to see more significant improvements in the future in the management and care of the disease.

Optimizing Platelet and Leucocyte-Rich Plasma as Biomaterials for Ophthalmic Applications: Impact of Centrifugation Speed.

Purpose: To assess the impact of varying centrifugation speeds on platelet and leucocyte-rich plasma (L-PRP) in liquid and gel form cellularity and growth factor concentrations for potential use against ocular surface disorders. Patients and Methods: L-PRP was collected from 16 healthy subjects using three different centrifugation speeds: 580, 1000, and 2000 g, each for 8 min at 25°C. Platelet and leukocyte counts were automatically evaluated. The concentrations of vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and transforming growth factor beta 1 (TGF-B1) were measured using enzyme-linked immunosorbent assays. L-PRP gel cellularity was assessed through hematoxylin-eosin and Masson's trichrome staining, categorized as moderate or abundant, and statistically analyzed. L-PRP gel membrane's chemical composition was analyzed using Fourier-transform infrared spectroscopy (FTIR), crystallization was investigated with X-ray diffraction (XRD), and ultrastructure was assessed using surface electron microscopy (SEM). Additionally, membrane degradation was evaluated over a 7-day period. Results: No significant differences in cellularity and growth factor concentrations among centrifugation speeds (p > 0.05) were found. Moderate cellularity predominated at 580 g and 2000 g, while abundant cellularity was observed at 1000 g. No significant differences were found techniques (p = 0.16). Masson's trichrome staining suggested the existence of abundant fibrin at 1000 g but without significant differences (p = 0.07). FTIR analysis exhibited the characteristic fibrin bands at all speeds, and XRD indicated a keratin-like pattern. SEM revealed greater porosity at 580 g and fibrin membrane degradation was lower at this speed (p = 0.0001). Conclusion: Centrifugation speed did not significantly affect growth factor concentration or cellularity in both liquid and gel L-PRP. Further studies should explore the impact of different separation techniques for L-PRP used in ophthalmic applications.

Customizable Collagen Vitrigel Membranes and Preliminary Results in Corneal Engineering.

Corneal opacities are a leading cause of visual impairment that affect 4.2 million people annually. The current treatment is corneal transplantation, which is limited by tissue donor shortages. Corneal engineering aims to develop membranes that function as scaffolds in corneal cell transplantation. Here, we describe a method for producing transplantable corneal constructs based on a collagen vitrigel (CVM) membrane and corneal endothelial cells (CECs). The CVMs were produced using increasing volumes of collagen type I: 1X (2.8 µL/mm2), 2X, and 3X. The vitrification process was performed at 40% relative humidity (RH) and 40 °C using a matryoshka-like system consisting of a shaking-oven harboring a desiccator with a saturated K2CO3 solution. The CVMs were characterized via SEM microscopy, cell adherence, FTIR, and manipulation in an ex vivo model. A pilot transplantation of the CECs/CVM construct in rabbits was also carried out. The thickness of the CVMs was 3.65-7.2 µm. The transparency was superior to a human cornea (92.6% = 1X; 94% = 2X; 89.21% = 3X). SEM microscopy showed a homogenous surface and laminar organization. The cell concentration seeded over the CVM increased threefold with no significant difference between 1X, 2X, and 3X (p = 0.323). The 2X-CVM was suitable for surgical manipulation in the ex vivo model. Constructs using the CECs/2X-CVM promoted corneal transparency restoration.

Phytochemical Profile and Antioxidant and Antiproliferative Activity of Sedum dendroideum on Pterygium Fibroblasts.

BACKGROUND: Sedum dendroideum has antioxidant effects that are beneficial for different diseases. We aimed to analyze the antiproliferative activity of S. dendroideum in human pterygium fibroblasts (HPFs). METHODS: HPFs were treated for 24 h with 0-1000 µg/mL of S. dendroideum lyophilized to analyze its effect on cell viability using the CellTiter assay. RNA from HPF treated with 250 µg/mL of S. dendroideum lyophilized was isolated, and the expression of VEGF and CTGF genes was evaluated by qPCR. A dermal fibroblast cell line (HDFa) was used as a healthy control. The total phenolic content, antioxidant activity, and chemical profile of S. dendroideum lyophilized were determined. RESULTS: Viability of HPF decreased after 24 h treatment of S. dendroideum in a dose-dependent manner. The expression of VEGF and CTGF significantly decreased (P < 0.01) in HPF treated with 250 µg/mL of S. dendroideum when compared with untreated HPF. The total phenolic concentration in the S. dendroideum lyophilized was 33.67 mg gallic acid equivalents (GAE)/g. Antioxidant activity was 384.49 mM Trolox equivalents/mL. The main phenolic compounds identified by HPLC analysis were the kaempferol-3-O-glycoside, kaempferol-3-O-rhamnoside, kaempferol-3-O-neohesperidoside-7-O-α-rhamnopyranoside, and kaempferol-3-O-glycoside-7-O-rhamnoside. CONCLUSIONS: S. dendroideum decreases the proliferation of HPF and the expression of VEGF and CTGF. The phenolic compound concentration, antioxidant activity, and phytochemical profile may play a role in these effects.

Experimental design of a culture approach for corneal endothelial cells of New Zealand white rabbit.

The harvesting of corneal endothelial cells (CEC) has received special attention due to its potential as a therapy for corneal blindness. The main challenges are related to the culture media formulation, cellular density at the primary isolation, and the number of passages in which CEC can retain their functional characteristics. To alternate different media formulations to harvest CEC has an impact on the cellular yield and morphology. Therefore, we analyzed four different sequences of growth factor-supplemented Stimulatory (S) and non-supplemented Quiescent (Q) media, upon passages to find the optimal S-Q culture sequence. We assessed cell yield, morphology, procollagen I production, Na+/K+-ATPase function, and the expression of ZO-1 and Na+/K+-ATPase. Our results show SQSQ and SQQQ sequences with a balance between an improved cell yield and hexagonal morphology rate. CEC cultured in the SQQQ sequence produced procollagen I, showed Na+/K+-ATPase function, and expression of ZO-1 and Na+/K+-ATPase. Our study sets a culture approach to guarantee CEC expansion, as well as functionality for their potential use in tissue engineering and in vivo analyses. Thus, the alternation of S and Q media improves CEC culture. SQQQ sequence demonstrated CEC proliferation and lower the cost implied in SQSQ sequences. We discarded the use of pituitary extract and ROCK inhibitors as essential for CEC proliferation.

Arresting proliferation improves the cell identity of corneal endothelial cells in the New Zealand rabbit.

Purpose: Corneal endothelium engineering aims to reduce the tissue shortage for corneal grafts. We investigated the impact of mitogenic and resting culture systems on the identity of corneal endothelial cells (CECs) for tissue engineering purposes. Methods: Rabbit CECs were cultured in growth factor-supplemented media (MitoM) until confluence. At the first passage, the CECs were divided into two populations: P1 remained cultured in MitoM, and P2 was cultured in a basal medium (RestM) for another passage. Morphologic changes in the CECs were analyzed, and RNA was isolated for transcriptome analysis. Quantitative PCR and immunocytochemistry validation of selected differentially expressed markers were performed. Results: The CECs in MitoM showed fibroblastic morphology, whereas the CECs in RestM exhibited polygonal morphology. Circularity analysis showed similar values in human (0.75±0.056), rabbit basal (before cultured; 0.77±0.063), and CECs in RestM (0.73±0.09), while MitoM showed lower circularities (0.41±0.19). Genes related to collagen type IV and the extracellular matrix, along with the adult CEC markers ATP1A1, ATP1B1, COL8A2, GPC4, and TJP1, were highly expressed in RestM. Conversely, the IL-6, F3, and ITGB3 genes and the non-adult CEC markers CD44, CNTN3, and CD166 were more expressed in MitoM. Overall, from the transcriptome, we identified 832 differentially expressed probes. A functional analysis of the 308 human annotated differentially expressed genes revealed around 13 functional clusters related to important biological terms, such as extracellular matrix, collagen type 4, immune responses, cell proliferation, and wound healing. Quantitative PCR and immunocytochemistry confirmed the overexpression of ATP1A1, TJP1, and GPC4 in CECs in RestM. Conclusions: The addition of a stabilization step during CEC culture improves the cells' morphology and molecular identity, which agrees with transcriptome data. This suggests that stabilization is useful for studying the plasticity of the corneal endothelium's morphology, and stabilization is proposed as a necessary step in corneal endothelium engineering.

Extracellular matrix and fibroblast injection produces pterygium-like lesion in rabbits.

BACKGROUND: Translational research to develop pharmaceutical and surgical treatments for pterygium requires a reliable and easy to produce animal model. Extracellular matrix and fibroblast are important components of pterygium. The aim of this study was to analyze the effect of the subconjunctival injection of fibroblast cells (NIH3T3 cell line) and exogenous extracellular matrix in rabbits in producing a pterygium-like lesion. METHODS: Six 3-month-old white New Zealand rabbits were injected with 20,000 NIH3T3 cells and 5 µL of Matrigel in the right conjunctiva, and with only 5 µL of Matrigel in the left conjunctiva. The eyes were photographed under a magnification of 16× using a 12-megapixel digital camera attached to the microscope on day 1, 3 and 7. Conjunctival vascularization was measured by analyzing images to measure red pixel saturation. Area of corneal and conjunctival fibrovascular tissue formation on the site of injection was assessed by analyzing the images on day 3 and 7 using area measurement software. Histopathologic characteristics were determined in the rabbit tissues and compared with a human primary pterygium. RESULTS: The two treatments promoted growth of conjunctival fibrovascular tissue at day 7. The red pixel saturation and area of fibrovascular tissue developed was significantly higher in right eyes (p < 0.05). Tissues from both treatments showed neovascularization in lesser extent to that observed in human pterygium. Acanthosis, stromal inflammation, and edema were found in tissues of both treatments. No elastosis was found in either treatment. CONCLUSIONS: Matrigel alone or in combination with NIH3T3 cells injected into the rabbits' conjunctiva can promote tissue growth with characteristics of human pterygium, including neovascularization, acanthosis, stromal inflammation, and edema. The combination of Matrigel with NIH3T3 cells seems to have an additive effect on the size and redness of the pterygium-like tissue developed.

Primary explant culture and collagen I substrate enhances corneal endothelial cell morphology.

OBJECTIVES: Corneal endothelial cell (CEC) isolation and harvest aim to produce engineered grafts to solve donor corneal tissue shortage. To yield high amounts of CEC maintaining morphological and molecular characteristics, several isolation and culture conditions are reported. Here, we combined direct explant culture, with three different coating conditions and a two-step media approach to compare confluence efficiency, morphology, and specific molecular markers expression. DATA DESCRIPTION: Confluence was reached after 2 weeks in the three coating conditions (Matrigel, collagen I, and in uncoated plates) using a two-step approach (proliferative medium without pituitary extract, followed by stabilizer basal medium). Na/K-ATPase and GPC4 markers were detected by immunocytochemistry while GPC4, CD200, and TJP1 by RT-PCR in the three CEC coating culture conditions. CEC in proliferative medium showed spindle morphology in the three conditions. Polygonal morphology was seen in CEC cultures using basal medium under uncoated and collagen I coated plates. CEC cultured in Matrigel-coated plates remained with spindle morphology in basal medium.

Extracellular matrix and fibroblast injection produces pterygium-like lesion in rabbits

BACKGROUND: Translational research to develop pharmaceutical and surgical treatments for pterygium requires a reliable and easy to produce animal model. Extracellular matrix and fibroblast are important components of pterygium. The aim of this study was to analyze the effect of the subconjunctival injection of fibroblast cells (NIH3T3 cell line) and exogenous extracellular matrix in rabbits in producing a pterygium-like lesion. METHODS: Six 3-month-old white New Zealand rabbits were injected with 20,000 NIH3T3 cells and 5 µL of Matrigel in the right conjunctiva, and with only 5 µL of Matrigel in the left conjunctiva. The eyes were photographed under a magnification of 16× using a 12-megapixel digital camera attached to the microscope on day 1,3 and 7. Conjunctival vascularization was measured by analyzing images to measure red pixel saturation. Area of corneal and conjunctival fibrovascular tissue formation on the site of injection was assessed by analyzing the images on day 3 and 7 using area measurement software. Histopathologic characteristics were determined in the rabbit tissues and compared with a human primary pterygium. RESULTS: The two treatments promoted growth of conjunctival fibrovascular tissue at day 7. The red pixel saturation and area of fibrovascular tissue developed was significantly higher in right eyes (p < 0.05). Tissues from both treatments showed neovascularization in lesser extent to that observed in human pterygium. Acanthosis, stromal inflammation, and edema were found in tissues of both treatments. No elastosis was found in either treatment. CONCLUSIONS: Matrigel alone or in combination with NIH3T3 cells injected into the rabbits' conjunctiva can promote tissue growth with characteristics of human pterygium, including neovascularization, acanthosis, stromal inflammation, and edema. The combination of Matrigel with NIH3T3 cells seems to have an additive effect on the size and redness of the pterygium-like tissue developed.

Diabetes mellitus en pacientes atendidos en el hospital universitario: Caracterización epidemiológica / Diabetes mellitus in patients of the Guayaquil university hospital: Epidemiological characterization

Introducción. La diabetes mellitus es una enfermedad metabólica que se caracteriza por la elevación de la glicemia en sangre; la misma que se ha observado un incremento en el número de pacientes adultos. Objetivo. Determinar la frecuencia de diabetes mellitus y los factores epidemiológicos importantes en los pacientes atendidos en el Hospital Universitario de Guayaquil, entre 2014-2015. Metodología. Diseño observacional de tipo transversal descriptivo en los pacientes de la consulta externa, en la que se utilizó un formulario de recolecciónde datos. Resultados. Se obtuvo diabetes mellitus no insulinodependiente el 45,16 %, diabetes insulinodependiente 39,87 %, diabetes mellitus no especificada 14,15 %; mayormente afectados el adulto intermedio (41 a 64 años de edad) con el 60,31 %; en el sexo femenino el 52,14 % comparado con el masculino con el 47,86 %; presentaron comorbilidad en aproximadamente 8,99 %, mayormente complicaciones de tipo circulatorias con el 10,75 %. Conclusiones. Existe mayormente diabetes mellitus no insulinodependiente, con predisposición en el sexo femenino, y en el grupo de adultas intermedias comparados con los masculinos; que quizá la tendencia presentada se deba a otros factores, como la falta de ejercicio, dieta no saludable, sobrepeso, historia familiar; fomentando la prevención y control de la diabetes mellitus mediante campañas integrales de información, educación y comunicación en todos los sectores de la sociedad.

Introduction. Diabetes Mellitus is a metabolic disease characterized by elevated blood glucose levels; the same has been observed an increase in the number of adult patients. Objective. To determine the frequency of Diabetes Mellitus and the important epidemiological factors in the patients treated at the University Hospital of Guayaquil between 2014-2015. Methodology. Observational design of descriptive transversal type; in the patients of the external consultation, in which a form of data collection was used. Results. Non-insulin dependent Diabetes Mellitus (NID) was obtained 45.16%, Insulin Dependent Diabetes (ID) 39.87%, Diabetes Mellitus Unspecified 14.15%; Mainly affected Intermediate Adult (41 to 64 years old) with 60.31%; In the female sex 52.14% compared to the male with 47.86%; Presented comorbidity in approximately 8.99%, mostly circulatory type complications with 10.75%. Conclusions. There is mainly Diabetes Mellitus Non-insulin dependent, predisposed in the female sex, and in the group of intermediate adults compared to the male; That the tendency presented may be due to other factors, such as lack of exercise, unhealthy diet, overweight, family history; Promoting the prevention and control of Diabetes Mellitus through comprehensive campaigns of information, education and communication in all sectors of society.

Caracterización clínica del dengue con signos de alarma y grave, en hospitales de Guayaquil / Clinical characterization of dengue with signs of alarm and severe, in hospitals of Guayaquil

Introducción. El dengue es una infección vírica transmitida por mosquitos cuya infección va desde síntomas leves que en ocasiones evolucionan hasta un cuadro clínico llamado dengue grave. Objetivo. Caracterizar clínicamente los casos con diagnóstico de dengue con signos de alarma y dengue grave entre las semanas epidemiológicas 19 al 26 del 2014 ingresados en el Hospital de Niños Francisco de Icaza Bustamante y en adultos del hospital de Infectología de la ciudad de Guayaquil. Metodología. No experimental de tipo descriptivo, comparativo, de corte transversal; donde se comparan sus características clínicas, antecedentes y valores en pruebas hemáticas. Resultados. La fiebre re registró en el total de los pacientes, que se acompañan en los niños con nausea o vómito, dolor abdominal, artralgias, mialgias, cefaleas, entre otros, con variedad sintomática, mientras que en los adultos concuerda con la definición de casos. En las pruebas de laboratorio se detectaron casos con hemoconcentración y plaquetopenia mayormente en adultos; TGO y TGP elevadas por encima del 70 % de los casos; presencia de IgM en aproximadamente 1 de cada 2 en niños y adultos al momento de su ingreso, con anticuerpos IgG en 2 de cada 3 niños que revela una previa exposición al dengue. Conclusiones. Mayor afectación en los niños con variedad sintomática, entre 3 a 7 días de evolución, acompañados de hemoconcentración y plaquetopenia; así como, leucopenia, linfocitosis, neutropenia y las enzimas hepáticas elevadas en gran número de casos. Las pruebas serológicas específicas para dengue demuestran la infección reciente y antecedentes de infección pasada.

Introduction. Dengue is a viral infection transmitted by mosquitoes; whose infection ranges from mild symptoms that sometimes evolve to a clinical condition called severe dengue. Objective. Clinically characterize the cases with diagnosis of dengue with signs of alarm and severe dengue among the epidemiological weeks 19 to 26 of 2014 admitted to the Hospital of children Francisco de Icaza Bustamante and in adults of the Hospital of Infectología of the city of Guayaquil. Methodology. Non-experimental descriptive, comparative, cross-sectional type; where their clinical characteristics, antecedents and values are compared in blood tests. Results. Fever had the total number of patients, who were accompanied in children with nausea or vomiting, abdominal pain, arthralgia, myalgias, headaches, among others, with symptomatic variety, while in adults it was consistent with the definition of cases. In laboratory tests cases with hemoconcentration and thrombocytopenia were detected mainly in adults; TGO and TGP elevated above 70% of the cases; Presence of IgM in approximately 1 in 2 in children and adults at the time of admission, with IgG antibodies in 2 of 3 children revealing a previous exposure to Dengue. Conclusions. Major affectation in children with symptomatic variety, between 3 and 7 days of evolution, accompanied by hemoconcentration and thrombocytopenia; As well as leukopenia, lymphocytosis, neutropenia and elevated liver enzymes in a large number of cases. Specific serological tests for dengue demonstrate recent infection and history of past infection.

Molecular Basis of Pterygium Development.

Pterygium pathogenesis is mainly related to UV light exposure. However, the exact mechanisms by which it is formed have not been elucidated. Clinical advances in surgical treatment use conjunctival autografts and amniotic membranes in combination with adjuvant therapies, including mitomycin C, ß-radiation, and 5-fluoroacil, to reduce recurrence. Several studies aim to unveil the molecular mechanisms underlying pterygium growth and proliferation. They demonstrate the role of different factors, such as viruses, oxidative stress, DNA methylation, apoptotic and oncogenic proteins, loss of heterozygosity, microsatellite instability, inflammatory mediators, extracellular matrix modulators, lymphangiogenesis, cell epithelial-mesenchymal transition, and alterations in cholesterol metabolism in pterygium development. Understanding the molecular basis of pterygium provides new potential therapeutic targets for its prevention and elimination. This review focuses on providing a broad overview of what is currently known regarding molecular mechanisms of pterygium pathogenesis.

Adult white New Zealand rabbit as suitable model for corneal endothelial engineering.

BACKGROUND: Corneal endothelium engineering is focused in producing transplantable cell sheets to overcome the shortage of corneal graft tissue donors for the treatment of corneal blindness. For this purpose, the use of a proper animal model plays a key role. Corneal parameters of White New Zealand rabbits such as endothelial cell density, central corneal thickness, and corneal diameter decrease with age, similarly as in humans. However, as opposed to humans, they retain the ability to restore their corneal endothelium after injury. Therefore, they are considered as an inappropriate corneal endothelial wound healing model. FINDINGS: Here we analyze the corneal endothelium mitotic ability of White New Zealand rabbits aged 3, 6, 12 and 18 months, 36 and 72 hours after thermal injury. The highest mitotic activity was observed in the 3-month rabbits 36 h after wounding. Rabbits of 12 months registered decreased mitotic activity and those of 18 months did not show mitotic activity 72 h after injury. CONCLUSIONS: These results propose that rabbits of 18 months represent a suitable model for human corneal endothelium engineering research.