RESUMEN
Tracking cell death in vivo can enable a better understanding of the biological mechanisms underlying tissue homeostasis and disease. Unfortunately, existing cell death labeling methods lack compatibility with in vivo applications or suffer from low sensitivity, poor tissue penetration, and limited temporal resolution. Here, we fluorescently labeled dead cells in vivo with Trypan Blue (TBlue) to detect single scattered dead cells or to generate whole-mount three-dimensional maps of large areas of necrotic tissue during organ regeneration. TBlue effectively marked different types of cell death, including necrosis induced by CCl4 intoxication in the liver, necrosis caused by ischemia-reperfusion in the skin, and apoptosis triggered by BAX overexpression in hepatocytes. Moreover, due to its short circulating lifespan in blood, TBlue labeling allowed in vivo "pulse and chase" tracking of two temporally spaced populations of dying hepatocytes in regenerating mouse livers. Additionally, upon treatment with cisplatin, TBlue labeled dead cancer cells in livers with cholangiocarcinoma and dead thymocytes due to chemotherapy-induced toxicity, showcasing its utility in assessing anticancer therapies in preclinical models. Thus, TBlue is a sensitive and selective cell death marker for in vivo applications, facilitating the understanding of the fundamental role of cell death in normal biological processes and its implications in disease.
Asunto(s)
Muerte Celular , Azul de Tripano , Animales , Ratones , Muerte Celular/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Hepatocitos/patología , Hepatocitos/metabolismo , Humanos , Neoplasias/patología , Ratones Endogámicos C57BL , Regeneración Hepática/efectos de los fármacos , Hígado/patología , Hígado/efectos de los fármacos , Rastreo Celular/métodos , Apoptosis/efectos de los fármacos , Imagenología Tridimensional , Regeneración/efectos de los fármacos , Necrosis , MasculinoRESUMEN
Amazonian strains of Cyathus spp. and Geastrum spp. were studied for the ability to discolor the trypan blue azo dye and reduce its toxicity. Discoloration of trypan blue dye (0.05%) was evaluated in solid and aqueous medium over different periods. The reduction of dye toxicity after treatment was assessed by seed germination and the development of lettuce seedlings (Lactuca sativa L.) and toxicity test in Artemia salina (L.) larvae. All evaluated strains showed the potential to reduce the color intensity of trypan blue dye. Cyathus strains reached 96% discoloration, and C. albinus and C. limbatus also reduced dye toxicity. Geastrum strains showed a high efficiency degree in color reduction, reaching 98% discoloration, however, the by-products generated during the process presented toxicity and require further investigation. For the first time, Amazonian strains of gasteroid fungi degrading trypan blue are reported, some even reducing its toxicity. Thus, making them promising sources of enzymes of interest to bioremediation scenarios involving synthetic dyes.
Asunto(s)
Basidiomycota , Azul de Tripano , Compuestos Azo/toxicidad , Compuestos Azo/metabolismo , Biodegradación Ambiental , Basidiomycota/metabolismo , Hongos , Colorantes/toxicidadRESUMEN
BACKGROUND: Parathyroid hormone-related peptide (PTHrP) plays a key role in the development and progression of many tumors. We found that in colorectal cancer (CRC) HCT116 cells, the binding of PTHrP to its receptor PTHR type 1 (PTHR1) activates events associated with an aggressive phenotype. In HCT116 cell xenografts, PTHrP modulates the expression of molecular markers linked to tumor progression. Empirical evidence suggests that the Met receptor is involved in the development and evolution of CRC. Based on these data, we hypothesized that the signaling pathway trigged by PTHrP could be involved in the transactivation of Met and consequently in the aggressive behavior of CRC cells. AIM: To elucidate the relationship among PTHR1, PTHrP, and Met in CRC models. METHODS: For in vitro assays, HCT116 and Caco-2 cells derived from human CRC were incubated in the absence or presence of PTHrP (1-34) (10-8 M). Where indicated, cells were pre-incubated with specific kinase inhibitors or dimethylsulfoxide, the vehicle of the inhibitors. The protein levels were evaluated by Western blot technique. Real-time polymerase chain reaction (RT-qPCR) was carried out to determine the changes in gene expression. Wound healing assay and morphological monitoring were performed to evaluate cell migration and changes related to the epithelial-mesenchymal transition (EMT), respectively. The number of viable HCT116 cells was counted by trypan blue dye exclusion test to evaluate the effects of irinotecan (CPT-11), oxaliplatin (OXA), or doxorubicin (DOXO) with or without PTHrP. For in vivo tests, HCT116 cell xenografts on 6-wk-old male N:NIH (S)_nu mice received daily intratumoral injections of PTHrP (40 µg/kg) in 100 µL phosphate-buffered saline (PBS) or the vehicle (PBS) as a control during 20 d. Humanitarian slaughter was carried out and the tumors were removed, weighed, and fixed in a 4% formaldehyde solution for subsequent treatment by immunoassays. To evaluate the expression of molecular markers in human tumor samples, we studied 23 specimens obtained from CRC patients which were treated at the Hospital Interzonal de Graves y Agudos Dr. José Penna (Bahía Blanca, Buenos Aires, Argentina) and the Hospital Provincial de Neuquén (Neuquén, Neuquén, Argentina) from January 1990 to December 2007. Seven cases with normal colorectal tissues were assigned to the control group. Tumor tissue samples and clinical histories of patients were analyzed. Paraffin-embedded blocks from primary tumors were reviewed by hematoxylin-eosin staining technique; subsequently, representative histological samples were selected from each patient. From each paraffin block, tumor sections were stained for immunohistochemical detection. The statistical significance of differences was analyzed using proper statistical analysis. The results were considered statistically significant at P < 0.05. RESULTS: By Western blot analysis and using total Met antibody, we found that PTHrP regulated Met expression in HCT116 cells but not in Caco-2 cells. In HCT116 cells, Met protein levels increased at 30 min (P < 0.01) and at 20 h (P < 0.01) whereas the levels diminished at 3 min (P < 0.05), 10 min (P < 0.01), and 1 h to 5 h (P < 0.01) of PTHrP treatment. Using an active Met antibody, we found that where the protein levels of total Met decreased (3 min, 10 min, and 60 min of PTHrP exposure), the status of phosphorylated/activated Met increased (P < 0.01) at the same time, suggesting that Met undergoes proteasomal degradation after its phosphorylation/activation by PTHrP. The increment of its protein level after these decreases (at 30 min and 20 h) suggests a modulation of Met expression by PTHrP in order to improve Met levels and this idea is supported by our observation that the cytokine increased Met mRNA levels at least at 15 min in HCT116 cells as revealed by RT-qPCR analysis (P < 0.05). We then proceeded to evaluate the signaling pathways that mediate the phosphorylation/ activation of Met induced by PTHrP in HCT116 cells. By Western blot technique, we observed that PP1, a specific inhibitor of the activation of the proto-oncogene protein tyrosine kinase Src, blocked the effect of PTHrP on Met phosphorylation (P < 0.05). Furthermore, the selective inhibition of the ERK 1/2 mitogen-activated protein kinase (ERK 1/2 MAPK) using PD98059 and the p38 MAPK using SB203580 diminished the effect of PTHrP on Met phosphorylation/activation (P < 0.05). Using SU11274, the specific inhibitor of Met activation, and trypan blue dye exclusion test, Western blot, wound healing assay, and morphological analysis with a microscope, we observed the reversal of cell events induced by PTHrP such as cell proliferation (P < 0.05), migration (P < 0.05), and the EMT program (P < 0.01) in HCT116 cells. Also, PTHrP favored the chemoresistance to CPT-11 (P < 0.001), OXA (P < 0.01), and DOXO (P < 0.01) through the Met pathway. Taken together, these findings suggest that Met activated by PTHrP participates in events associated with the aggressive phenotype of CRC cells. By immunohistochemical analysis, we found that PTHrP in HCT116 cell xenografts enhanced the protein expression of Met (0.190 ± 0.014) compared to tumors from control mice (0.110 ± 0.012; P < 0.05) and of its own receptor (2.27 ± 0.20) compared to tumors from control mice (1.98 ± 0.14; P < 0.01). Finally, assuming that the changes in the expression of PTHrP and its receptor are directly correlated, we investigated the expression of both Met and PTHR1 in biopsies of CRC patients by immunohistochemical analysis. Comparing histologically differentiated tumors with respect to those less differentiated, we found that the labeling intensity for Met and PTHR1 increased and diminished in a gradual manner, respectively (P < 0.05). CONCLUSION: PTHrP acts through the Met pathway in CRC cells and regulates Met expression in a CRC animal model. More basic and clinical studies are needed to further evaluate the PTHrP/Met relationship.
Asunto(s)
Neoplasias Colorrectales , Proteína Relacionada con la Hormona Paratiroidea , Animales , Células CACO-2 , Movimiento Celular , Proliferación Celular , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Humanos , Irinotecán , Masculino , Ratones , Proteína Relacionada con la Hormona Paratiroidea/genética , Proteína Relacionada con la Hormona Paratiroidea/metabolismo , Proteína Relacionada con la Hormona Paratiroidea/farmacología , Azul de Tripano/farmacologíaRESUMEN
Acacia modesta (AM) and Opuntia monocantha (OM) are distributed in Pakistan, Afghanistan and India. Both of these plants have different pharmacological properties. This study was designed to evaluate anticancer potential of Acacia modesta (AM) and Opuntia monocantha (OM). Liver cancer cell line HepG2 was used for assessment of anticancer activity. For the evaluation of anti-proliferative effects, cell viability and cell death in all groups of cells were evaluated via MTT, crystal violet and trypan blue assays. For the evaluation of apoptosis ELISA of p53 performed. Furthermore, LDH assay to find out the ability of malignant cells to metabolize pyruvate to lactate and antioxidant enzymes activity (GSH, CAT and SOD) at the end HPLC was performed to find active compound of AM and OM. Cytotoxicity (MTT), Viability assays (trypan blue, crystal viability, MUSE analysis) showed more dead, less live cells in plant treated groups with increase of concentration. Scratch assay for the anti-migratory effect of these plants showed treated groups have not ability to heal scratch/wound. ELISA of p53 for cellular apoptosis showed more release of p53 in treated groups. Antioxidant assay via glutathione (GSH), superoxide dismutase (SOD), catalase (CAT) showed less anti-oxidative potential in treated cancer groups. LDH assay showed more lactate dehydrogenase release in treated groups compared with untreated. HPLC analysis showed the presence of phytochemicals such as steroids, alkaloids, phenols, flavonoids, saponins, tannins, anthraquinone and amino acids in AM and OM plant extracts. Based on all these findings, it can be concluded that ethanolic extracts of Acacia modesta and Opuntia monocantha have promising anti-cancer potential.
Asunto(s)
Acacia , Neoplasias Hepáticas , Opuntia , Extractos Vegetales , Acacia/química , Antioxidantes/farmacología , Células Hep G2 , Humanos , Opuntia/química , Extractos Vegetales/farmacología , Superóxido Dismutasa/metabolismo , Azul de Tripano , Proteína p53 Supresora de TumorRESUMEN
Background: The endothelium is a layer fundamental to maintaining corneal transparency. In ophthalmology, sheep eyes have been used as a model in research related to corneal transplantation. Different techniques have been used to evaluate the corneal endothelium. Concerning vital dyes, corneal endothelial cell analyses have not yet been studied in ovines. The purpose of the present study was to evaluate the morphology of endothelial cells from different regions of the cornea of sheep after staining with alizarin red and trypan blue using an optical microscope. Materials, Methods & Results: Twenty healthy eyes of 10 male sheep obtained from a licensed commercial slaughterhouse were studied. The study was approved by the Research Committee of the Faculty of Veterinary at UFRGS and followed the ethical standards of the Association for Research in Vision and Ophthalmology (ARVO). Immediately after the slaughter, the eyes were enucleated and underwent eye examination. The corneal endothelium was stained with trypan blue and alizarin red and examined and photographed using an optical microscope. The central, superior, inferior, nasal and temporal areas of the cornea were evaluated for cell morphology. Data were compared by t-tests. Differences were considered statistically significant at P < 0.05. Immediately after staining the corneal endothelium, it was possible to examine with an optical microscope, obtain images and analyse the shape of endothelial cells from all regions of the sheep cornea. Polygonal, uniform and continuous cells were observed in all samples studied. Considering all the corneas analysed, cells with 6 sides (75.11%), 5 sides (12.76%) and 4 sides (12.12%) were found. In the central region of the cornea 75.91% of cells with 6 sides, 12.6% of cells with 5 sides and 11.48% with 7 sides were found. In the superior region of the cornea 76.07% of cells with 6 sides, 13.25% with 5 sides and 10.68% with 7 sides were found. In the lower region were found 74.72% of cells with 6 sides, 13% with 5 sides and 12.27% with 7 sides. In the temporal region, 74.14% were 6-sided cells, 11.42% had 5 sides, and 14.43% had 7 sides. Furthermore, in the nasal region, 74.72% of the cells had 6 sides, 13.54% had 5 sides, and 11.73% had 7 sides. No significant differences were found between cell morphology in all corneal regions evaluated. In addition, no significant difference was found when comparing the right eye with the left eye. Discussion: Different methods are used for the analysis of corneal endothelium. For ex vivo research optical microscopy after endothelial staining is an alternative low-cost technique that allows the analysis of all regions of the cornea. Quantitative analyses must characterise the endothelial parameters of the different species. The analysis of the morphology of corneal endothelium with an optic microscope after staining with alizarin red has been described as an effective, rapid and cost-efficient method, since this dye blends with the borated cells, allowing identification. In the present study, using optical microscopy and coloration with alizarin red it was possible to explore and obtain images of the ovine endothelium of all regions of the cornea. In the current study, the endothelium had a predominance of cells will 6 sides in all regions studied. This study allowed us to obtain images of the endothelium as well as quantitative data on the morphology of the different regions of the sheep cornea. This study demonstrated that morphology did not differ between the central and peripheral regions. The findings of this study represent a further source of reproducible data that should be considered when using sheep cornea as ex vivo model for experimental research.
Asunto(s)
Animales , Azul de Tripano/uso terapéutico , Ovinos , Endotelio Corneal/anatomía & histología , Indicadores y Reactivos/administración & dosificación , Microscopía/veterinariaRESUMEN
ABSTRACT: Chromovitrectomy, the intraocular application of dyes to assist visualization of preretinal tissues during vitreoretinal surgery, was introduced to avoid ocular complications related to internal limiting membrane peeling, inadequate removal of the vitreous, and incomplete removal of epiretinal membranes. Since 2000, chromovitrectomy has become a popular approach among vitreoretinal specialists. The first vital dye used in chromovitrectomy, indocyanine green, facilitated identification of the fine and transparent internal limiting membrane. Following indocyanine green, trypan blue was introduced to identify epiretinal membranes, and triamcinolone acetonide stained the vitreous well. Recently, additional natural dyes such as lutein and anthocyanin from the açaí fruit have been proposed for intraocular application during vitrectomy. The main goal of this review was to study the role of vital stains in chromovitrectomy and report the latest findings in the literature.
Asunto(s)
Colorantes/administración & dosificación , Vitrectomía/métodos , Cirugía Vitreorretiniana/métodos , Cuerpo Vítreo/cirugía , Membrana Epirretinal/metabolismo , Humanos , Verde de Indocianina/administración & dosificación , Procedimientos Quirúrgicos Oftalmológicos , Coloración y Etiquetado/métodos , Azul de Tripano/administración & dosificaciónRESUMEN
Toxocara spp. are responsible for causing toxocariasis, a zoonotic disease of global significance. In some countries of South America, toxocariasis is considered the most prevalent human helminthic infection. The objective of this study was to evaluate LIVE/DEAD® Viability/Cytotoxicity kit as an alternative method to analyze the viability of Toxacara cati larvae. Two control groups were used to confirm the usage of this methodology: 100 untreated T. cati larvae as a negative control (G1) and 100 T. cati larvae killed by thermal shock as a positive control (G2). Subsequently, the viability of T. cati larvae was assessed by the exclusion of the trypan blue dye and by LIVE/DEAD® Viability/Cytotoxicity kit, as well as observation of motility and morphology. In order to confirm the larvicidal effect, T. cati larvae G1 and G2 were inoculated in mice to evaluate their progression in vivo. As expected, G1 showed negative staining by Trypan blue and was stained green by LIVE/DEAD® Viability/Cytotoxicity kit in all the exposure periods. Moreover, G1 presented 100% of relative motility (RM) (score of 5). G2 group was stained blue by Trypan blue and red by LIVE/DEAD® Viability/Cytotoxicity kit, and had 0% RM (score zero) in 24 h of incubation period. In mice, G2 was not viable and, therefore, was not able to infect the animals. In mice inoculated with G1, however, larvae were recovered from all the evaluated organs, except eyes. These results demonstrate that the viability of T. cati larvae was accurately obtained by the LIVE/DEAD® Viability/Cytotoxicity kit, making it an alternative method for viability evaluation.
Asunto(s)
Toxocara/crecimiento & desarrollo , Análisis de Varianza , Animales , Membrana Celular/fisiología , Supervivencia Celular , Perros , Femenino , Larva/citología , Ratones , Ratones Endogámicos BALB C , Coloración y Etiquetado , Toxocara/citología , Toxocara/fisiología , Toxocariasis/parasitología , Azul de TripanoRESUMEN
Toxocara spp. are responsible for causing toxocariasis, a zoonotic disease of global importance, which is difficult to treat as the available drugs have moderate efficacy in the clinical resolution of the disease. A promising alternative to the existing drugs is Propolis, which is known for having biological and pharmacological properties such as antiparasitic, antioxidant, and antitumor activities. In this study, we report the in vitro anthelmintic activity of essential oil from Brazilian Red Propolis (EOP) against larvae of Toxocara cati. Approximately 100 larvae per well were cultivated in microplates containing RPMI-1640 medium and incubated in the presence of EOP (18.75, 37.5, 75, 150, 300 and 600⯵g/mL) to determine the Minimum Inhibitory Concentration (MIC) and IC50 (concentration required to inhibit 50% of the population) values. Then, T. cati larvae treated with the MIC of EOP were inoculated in mice to evaluate their progression in vivo. A concentration of 600⯵g/mL of EOP showed 100% larvicidal activity after exposure for 48â¯h, while 300⯵g/mL represented the IC50 and CC50. The anthelmintic activity of EOP was confirmed by the inability of the treated T. cati larvae to infect the mice. Our findings demonstrate the potential of EOP as an anthelmintic.
Asunto(s)
Antihelmínticos/farmacología , Aceites Volátiles/farmacología , Própolis/química , Toxocara/efectos de los fármacos , Animales , Antihelmínticos/aislamiento & purificación , Antihelmínticos/toxicidad , Células CHO , Colorantes , Cricetinae , Cricetulus , Femenino , Concentración 50 Inhibidora , Cinética , Larva/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Movimiento/efectos de los fármacos , Aceites Volátiles/aislamiento & purificación , Aceites Volátiles/toxicidad , Toxocara/fisiología , Azul de TripanoRESUMEN
INTRODUCTION: It is hypothesized that increased macrophage migration inhibitory factor (MIF) expression may contribute to diabetic nephropathy (DN) pathogenesis. The aim of the present study was to investigate the renal effects of MIF inhibition in a diabetic experimental model. METHODS: Eighteen male Wistar rats (230 ± 20 g) were divided into three groups: 1) control, 2) diabetic (STZ, 50 mg/kg, dissolved in saline, ip), 3) diabetic + MIF antagonist (p425, 1 mg/kg per day, ip, on the 21th day, for 21 consecutive days). The treatment started since we founwd a significant increase in urine albumin excretion (UAE) rate in the diabetic rats in comparison with the control rats. The rats were kept individually in metabolic cages (8 AM-2 PM) and urine samples were collected in the 21 and 42th day. At the end, blood and tissue samples were collected for biochemical (BS, UPE, urine GAG, BUN, Cr, Na, and K) and histological analyses. RESULTS: The results of this study showed that MIF antagonist (p425) significantly decreased urine protein and GAG excretion, urine protein/creatinine ratio, and serum BUN and Cr in the streptozotocin-induced DN in the rats. Pathological changes were significantly alleviated in the MIF antagonist (p425)-administered DN rats. CONCLUSION: Collectively, these data suggested that MIF antagonist (p425) was able to protect against functional and histopathological injury in the DN.
Asunto(s)
Diabetes Mellitus Experimental/patología , Nefropatías Diabéticas/tratamiento farmacológico , Oxidorreductasas Intramoleculares/antagonistas & inhibidores , Factores Inhibidores de la Migración de Macrófagos/antagonistas & inhibidores , Sustancias Protectoras/farmacología , Sustancias Protectoras/uso terapéutico , Azul de Tripano/farmacología , Azul de Tripano/uso terapéutico , Albuminuria/tratamiento farmacológico , Animales , Glucemia , Creatinina/sangre , Creatinina/orina , Diabetes Mellitus Experimental/sangre , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/orina , Nefropatías Diabéticas/sangre , Nefropatías Diabéticas/patología , Nefropatías Diabéticas/orina , Modelos Animales de Enfermedad , Glicosaminoglicanos/orina , Riñón/patología , Activación de Macrófagos , Masculino , Ratas , Ratas Wistar , Estreptozocina/farmacologíaRESUMEN
PURPOSE: Phacoemulsification has been cited as a possible cause of bleb failure in eyes with prior trabeculectomy. No method has been developed to directly evaluate the risk of bleb failure after phacoemulsification. We investigate the use of trypan blue during cataract surgery in the setting of a preexisting trabeculectomy to evaluate the functional status of the bleb and predict postoperative bleb function. MATERIALS AND METHODS: In total, 14 patients contributing 1 eye each with a history of prior trabeculectomy with mitomycin C undergoing phacoemulsification with intraocular lens implantation were enrolled in this prospective, nonrandomized clinical trial. At the time of phacoemulsification, trypan blue was instilled into the anterior chamber before capsulorhexis creation. Staining of the bleb was grouped as being mild or diffuse using intraoperative photographs. These eyes were followed for 1 year postoperatively and evaluated for intraocular pressure (IOP) control. RESULTS: The change in IOP was not significantly different between the 2 groups (P=0.14). A trend towards greater need for IOP-lowering medications was noted (P<0.10) in eyes with mild bleb staining. No statistically significant difference in rates of decreased bleb function was noted at 1-year follow-up after phacoemulsification. CONCLUSION: The intensity of bleb staining with trypan blue during phacoemulsification is not associated with changes in IOP postoperatively. A trend towards decreased need for IOP-lowering medications was noted in eyes with diffuse bleb staining at 1 year after cataract surgery.
Asunto(s)
Colorantes/administración & dosificación , Facoemulsificación/métodos , Estructuras Creadas Quirúrgicamente/fisiología , Azul de Tripano/administración & dosificación , Anciano , Anciano de 80 o más Años , Alquilantes/administración & dosificación , Terapia Combinada , Conjuntiva/efectos de los fármacos , Femenino , Estudios de Seguimiento , Humanos , Presión Intraocular/fisiología , Implantación de Lentes Intraoculares , Masculino , Mitomicina/administración & dosificación , Complicaciones Posoperatorias , Estudios Prospectivos , Coloración y Etiquetado , Tonometría Ocular , Trabeculectomía/métodosRESUMEN
Currently, there is no effective technique to evaluate the quality of oocytes in fish farming in apractical and affordable way. The cell membrane integrity test with the vital dye trypan blue (TB)could be an option. In this study, Colossoma macropomum and Brycon amazonicus oocytes were exposed to different TB concentrations seeking to verify a possible relationship between the results of staining tests and reproductive rates. Oocytes were exposed to concentrations of 0.05, 0.04, 0.03, 0.02 and 0.01% TB for 1 minute and subsequently were evaluated under a stereomicroscope. The percentage of unstained (viable) oocytes from each sample was correlated with fertilization and hatching rates using a linear regression (P>0.05). We observed a weak correlation between the results of the staining tests and the fertilization and hatching rates in both species. TB integrity tests were not effective in predicting spawning viability in C. macropomum and B. amazonicus.
Atualmente não existem técnicas efetivas para avaliar a qualidade de oócitos na piscicultura deforma prática e acessível. O teste de integridade da membrana celular com corante vital azul detripan (AT) surge como uma alternativa. Nesse estudo, oócitos de Colossoma macropomum e Bryconamazonicus foram expostos a diferentes concentrações de AT, buscando verificar uma possível relação entre os resultados dos testes de coloração e as taxas reprodutivas. Oócitos foram expostos a concentrações de 0,05; 0,04; 0,03; 0,02 e 0,01% de AT durante 1 minuto e, posteriormente avaliados em estereomicroscópio. A porcentagem de oócitos não corados (intactos) de cada amostra foicorrelacionada com as taxas de fertilização e eclosão utilizando análise de regressão linear (P>0,05). Fraca correlação entre resultados dos tratamentos e as taxas de fertilização e eclosão nas duas espécies foi observada. Os testes de integridade com AT foram ineficazes em predizer o sucesso da desova em C. macropomum e B. amazonicus
Asunto(s)
Animales , Characidae , Células Germinativas , Oocitos , Azul de Tripano/análisis , Coloración y Etiquetado , OviposiciónRESUMEN
Currently, there is no effective technique to evaluate the quality of oocytes in fish farming in apractical and affordable way. The cell membrane integrity test with the vital dye trypan blue (TB)could be an option. In this study, Colossoma macropomum and Brycon amazonicus oocytes were exposed to different TB concentrations seeking to verify a possible relationship between the results of staining tests and reproductive rates. Oocytes were exposed to concentrations of 0.05, 0.04, 0.03, 0.02 and 0.01% TB for 1 minute and subsequently were evaluated under a stereomicroscope. The percentage of unstained (viable) oocytes from each sample was correlated with fertilization and hatching rates using a linear regression (P>0.05). We observed a weak correlation between the results of the staining tests and the fertilization and hatching rates in both species. TB integrity tests were not effective in predicting spawning viability in C. macropomum and B. amazonicus.(AU)
Atualmente não existem técnicas efetivas para avaliar a qualidade de oócitos na piscicultura deforma prática e acessível. O teste de integridade da membrana celular com corante vital azul detripan (AT) surge como uma alternativa. Nesse estudo, oócitos de Colossoma macropomum e Bryconamazonicus foram expostos a diferentes concentrações de AT, buscando verificar uma possível relação entre os resultados dos testes de coloração e as taxas reprodutivas. Oócitos foram expostos a concentrações de 0,05; 0,04; 0,03; 0,02 e 0,01% de AT durante 1 minuto e, posteriormente avaliados em estereomicroscópio. A porcentagem de oócitos não corados (intactos) de cada amostra foicorrelacionada com as taxas de fertilização e eclosão utilizando análise de regressão linear (P>0,05). Fraca correlação entre resultados dos tratamentos e as taxas de fertilização e eclosão nas duas espécies foi observada. Os testes de integridade com AT foram ineficazes em predizer o sucesso da desova em C. macropomum e B. amazonicus(AU)
Asunto(s)
Animales , Characidae , Oocitos , Células Germinativas , Azul de Tripano/análisis , Coloración y Etiquetado , OviposiciónRESUMEN
ANTECEDENTES: Las tinciones capsulares constituyen uno de los mayores avances en la cirugía oftálmica al permitir la tinción de la cápsula anterior del cristalino en las cirugías de catarata. El azul de tripán se utiliza en los casos en los que no se visualiza de forma adecuada la cápsula anterior del cristalino cuando el reflejo rojo es pobre o nulo. Diferentes estudios señalan que el azul de tripán es un colorante eficaz y seguro para la tinción de la cápsula anterior del cristalino. METODOLOGIA: En la presente revisión se evaluó las diferencias en la efectividad entre las distintas concentraciones del azul del tripán. RESULTADOS: Los resultados muestran que el azul de tripán es una coloración segura y útil para la tinción de la cápsula anterior del cristalino. CONCLUSION: El azul de tripán tiene una amplia ventana terapéutica que permite ser utilizado a distintas concentraciones (0.0125%, 0.06%, 0.1%, 0.4% y 0.6%). Sin embargo, un estudio indicó que la concentración efectiva más baja para teñir el cristalino fue la de 0.1%.
Asunto(s)
Humanos , Extracción de Catarata/métodos , Cápsula del Cristalino/efectos de los fármacos , Azul de Tripano/administración & dosificación , Análisis Costo-Beneficio , Evaluación de la Tecnología BiomédicaRESUMEN
Even with the advances of conventional treatment techniques, the nervous system cancer prognosis is still not favorable to the patient which makes alternative therapies needed to be studied. Photodynamic therapy (PDT) is presented as a promising therapy, which employs a photosensitive (PS) agent, light wavelength suitable for the PS agent, and molecular oxygen, producing reactive oxygen species in order to induce cell death. The aim of this study is to observe the PDT action in gliosarcoma cell using a chlorin (Photodithazine, PDZ). The experiments were done with 9L/lacZ lineage cells, grown in a DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin solution and put in a culture chamber at 37 °C with an atmosphere of 5% CO2. The PS agent used was the PDZ to an LED light source device (Biopdi/IRRAD-LED 660) in the 660-nm region. The location of the PS agent was analyzed by fluorescence microscopy, and cell viability was analyzed by MTT assay (mitochondrial activity), exclusion by trypan blue (cell viability), and morphological examination through an optical microscope (Leica MD 2500). In the analysis of the experiments with PDZ, there was 100% cell death at different concentrations and clear morphological differences in groups with and without treatment. Furthermore, it was observed that the photodithazine has been focused on all nuclear and cytoplasmic extension; however, it cannot be said for sure whether the location is in the inside core region or on the plasma membrane. In general, the PDZ showed a promising photosensitive agent in PDT for the use of gliosarcoma.
Asunto(s)
Gliosarcoma/patología , Glucosamina/análogos & derivados , Fotoquimioterapia/métodos , Muerte Celular/efectos de los fármacos , Muerte Celular/efectos de la radiación , Línea Celular Tumoral , Forma de la Célula/efectos de los fármacos , Forma de la Célula/efectos de la radiación , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Glucosamina/farmacología , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/efectos de la radiación , Fármacos Fotosensibilizantes/farmacología , Azul de Tripano/metabolismoRESUMEN
The morpho-functional nature of the plasma membrane assists to transport nutrients into the interior of spermatozoa, which requires the physical integrity of the membrane. This present study aims to compare three different dyes used to determine the integrity of the plasma membrane of bovine spermatozoa after freezing. The dyes tested were eosin 3% (p/v), trypan blue 0.4% (p/v), and propidium iodide (1.5 mM). Two sample groups were formed for the research. In the first group, one ejaculate was collected from each 11 animals, and in the second group, ten ejaculates were obtained from one animal. The fresh semen was evaluated using microscopy. The post-freezing samples were assessed using microscopy and flow cytometry. The results are presented in the form of average viability percentages ± standard deviations. The values membrane integrity, for the dyes eosin, trypan blue, and propidium iodide were 56.8±8.3%; 56.1±7.9%; 54.6±9.1%; respectively. There were no significant differences between the groups (p≥0.001). In comparative analysis of the techniques, flow cytometry enabled a greater number of cell evaluations, compared to phase contrast microscopy, with less variability and faster sample evaluation, hence adding value to the microscopy tests.
O aspecto morfo-funcional da membrana plasmática facilita o transporte de nutrientes para o interior do espermatozoide, o que está ligado à integridade física da membrana plasmática. O presente trabalho objetivou comparar três diferentes corantes na avaliação da integridade física da membrana plasmática do espermatozoide bovino, pós-congelamento. Utilizando os corantes eosina 3% (p/v), azul de tripan 0,4% (p/v) e iodeto de propídio (1,5mM). Foram formados dois grupos para pesquisa. O primeiro, um ejaculado de cada 11 animais e um segundo grupo de dez ejaculados de um único animal. Foi realizada avaliação microscópica do sêmen fresco (motilidade total, motilidade progressiva, vigor e integridade da membrana plasmática). Das amostras congeladas foram avaliados todos os parâmetros descritos anteriormente, além da técnica de eosina, azul de tripan, e iodeto de propídio. Os resultados estão apresentados como média de porcentagem ± desvio padrão. Os valores para a integridade de membrana, utilizando os corantes eosina, azul de tripan, e iodeto de propídio, foram 56.8±8.3%; 56.1±7.9%; 54.6±9.1%, respectivamente. Não há diferença significativa entre os grupos (p≥0.001). Na análise comparativa das técnicas podemos observar que a citometria de fluxo, possibilita um número maior de avaliação celular, quando comparado à técnica de microscopia de campo claro, possibilitando uma menor variabilidade e maior rapidez na avaliação da amostra.
Asunto(s)
Animales , Bovinos , Eosina Amarillenta-(YS) , Azul de Tripano , Colorantes/análisis , Espermatozoides , Membrana Celular , Propidio , Criopreservación/veterinaria , Motilidad EspermáticaRESUMEN
The morpho-functional nature of the plasma membrane assists to transport nutrients into the interior of spermatozoa, which requires the physical integrity of the membrane. This present study aims to compare three different dyes used to determine the integrity of the plasma membrane of bovine spermatozoa after freezing. The dyes tested were eosin 3% (p/v), trypan blue 0.4% (p/v), and propidium iodide (1.5 mM). Two sample groups were formed for the research. In the first group, one ejaculate was collected from each 11 animals, and in the second group, ten ejaculates were obtained from one animal. The fresh semen was evaluated using microscopy. The post-freezing samples were assessed using microscopy and flow cytometry. The results are presented in the form of average viability percentages ± standard deviations. The values membrane integrity, for the dyes eosin, trypan blue, and propidium iodide were 56.8±8.3%; 56.1±7.9%; 54.6±9.1%; respectively. There were no significant differences between the groups (p≥0.001). In comparative analysis of the techniques, flow cytometry enabled a greater number of cell evaluations, compared to phase contrast microscopy, with less variability and faster sample evaluation, hence adding value to the microscopy tests.(AU)
O aspecto morfo-funcional da membrana plasmática facilita o transporte de nutrientes para o interior do espermatozoide, o que está ligado à integridade física da membrana plasmática. O presente trabalho objetivou comparar três diferentes corantes na avaliação da integridade física da membrana plasmática do espermatozoide bovino, pós-congelamento. Utilizando os corantes eosina 3% (p/v), azul de tripan 0,4% (p/v) e iodeto de propídio (1,5mM). Foram formados dois grupos para pesquisa. O primeiro, um ejaculado de cada 11 animais e um segundo grupo de dez ejaculados de um único animal. Foi realizada avaliação microscópica do sêmen fresco (motilidade total, motilidade progressiva, vigor e integridade da membrana plasmática). Das amostras congeladas foram avaliados todos os parâmetros descritos anteriormente, além da técnica de eosina, azul de tripan, e iodeto de propídio. Os resultados estão apresentados como média de porcentagem ± desvio padrão. Os valores para a integridade de membrana, utilizando os corantes eosina, azul de tripan, e iodeto de propídio, foram 56.8±8.3%; 56.1±7.9%; 54.6±9.1%, respectivamente. Não há diferença significativa entre os grupos (p≥0.001). Na análise comparativa das técnicas podemos observar que a citometria de fluxo, possibilita um número maior de avaliação celular, quando comparado à técnica de microscopia de campo claro, possibilitando uma menor variabilidade e maior rapidez na avaliação da amostra.(AU)
Asunto(s)
Animales , Bovinos , Membrana Celular , Eosina Amarillenta-(YS) , Azul de Tripano , Propidio , Colorantes/análisis , Espermatozoides , Criopreservación/veterinaria , Motilidad EspermáticaRESUMEN
Atualmente, a cápsula anterior e o epitélio da lente tem sido cada vez mais estudados, com o intuito de reduzir as possíveis complicações do pós-operatório da remoção da catarata, tal como a opacidade da cápsula posterior, alteração ocasionada principalmente pela diferenciação e migração das células do epitélio lenticular para a cápsula posterior da lente. O objetivo deste estudo foi analisar a composição molecular da cápsula anterior da lente pela técnica histoquímica de PAS (avaliação de proteoglicanos) e picrosirius red (avaliação de colágeno IV), em cães idosos com catarata diabética e não diabética do tipo hipermadura, submetidos ao uso ou não de azul de tripano a 0,1 % durante a facoemulsificação. Vinte e sete cães foram estudados, incluindo 21 fêmeas e 6 machos, de 8 a 12 anos de idade (média = 9,6 anos), de diversas raças e divididos em 2 grupos: GC (catarata hipermadura) e GCD (catarata diabética). Os resultados das análises realizadas mostraram que ambas as amostras, tanto as provenientes das cataratas hipermaduras, quanto das diabéticas, apresentam semelhante composição molecular de proteoglicanos e colágeno IV e isto independente da utilização de azul de tripano a 0,1 %. Conclui-se, portanto, que se os resultados obtidos forem decorrentes de alterações provocadas pelo rápido metabolismo da catarata diabética e pela cronicidade da catarata hipermadura sugere-se que o comprometimento da estrutura capsular seja de intensidade equivalente e, por consequência, que isto também possa prejudicar o metabolismo das células do epitélio anterior da lente, diminuindo assim a incidência da opacidade da cápsula posterior de cães com catarata diabética e hipermadura submetidos à facoemulsificação.(AU)
Nowadays, the anterior lens capsule and its epithelium have been being frequently studied aiming to reduce the incidence of posterior lens capsule opacity, a complication that frequently occurs after surgical removal of cataracts, due to epithelium cells differentiation and migration to the posterior pole. The objective of this study was to evaluate by histochemistry (PAS and picrosirius red) analysis two important molecular components of the anterior lens capsule (proteoglycans and type IV collagen) in older diabetic and non-diabetic dogs, with diabetic and hypermature cataracts, after phacoemulsification surgery utilizing 0.1% trypan blue or not. Twenty seven dogs, including 21 female and 6 male dogs, with ages varying from 8 to 12 years old (mean = 9.6 yo) of different breeds were studied. The animals were divided into 2 groups: GC (hypermature cataracts) and GCD (diabetic cataracts). Results showed that, besides their different pathophysiologies, both types of capsules studied (diabetic and hypermature ones) presented the same molecular composition of proteoglycans and type IV collagen, since no statistical significant differences were observed. In addition, 0.1% trypan blue was not capable to induce any other evident alteration for the samples. In conclusion, our findings suggest that, if the results consist in alteration induced by the aggressive metabolism of the diabetic cataract or the chronicity of the hypermature one, it is of the same intensity and independent of the use of 0.1% trypan blue. It is also possible to suggest that this alteration must be capable to compromise lens epithelium cell metabolism, which should probably favour future lens posterior capsule studies.(AU)
Asunto(s)
Animales , Perros , Cápsula Anterior del Cristalino/patología , Catarata/complicaciones , Catarata/veterinaria , Diabetes Mellitus/veterinaria , Cápsula Posterior del Cristalino/cirugía , Colágeno Tipo IV/análisis , Facoemulsificación/veterinaria , Proteoglicanos/análisis , Azul de TripanoRESUMEN
Atualmente, a cápsula anterior e o epitélio da lente tem sido cada vez mais estudados, com o intuito de reduzir as possíveis complicações do pós-operatório da remoção da catarata, tal como a opacidade da cápsula posterior, alteração ocasionada principalmente pela diferenciação e migração das células do epitélio lenticular para a cápsula posterior da lente. O objetivo deste estudo foi analisar a composição molecular da cápsula anterior da lente pela técnica histoquímica de PAS (avaliação de proteoglicanos) e picrosirius red (avaliação de colágeno IV), em cães idosos com catarata diabética e não diabética do tipo hipermadura, submetidos ao uso ou não de azul de tripano a 0,1 % durante a facoemulsificação. Vinte e sete cães foram estudados, incluindo 21 fêmeas e 6 machos, de 8 a 12 anos de idade (média = 9,6 anos), de diversas raças e divididos em 2 grupos: GC (catarata hipermadura) e GCD (catarata diabética). Os resultados das análises realizadas mostraram que ambas as amostras, tanto as provenientes das cataratas hipermaduras, quanto das diabéticas, apresentam semelhante composição molecular de proteoglicanos e colágeno IV e isto independente da utilização de azul de tripano a 0,1 %. Conclui-se, portanto, que se os resultados obtidos forem decorrentes de alterações provocadas pelo rápido metabolismo da catarata diabética e pela cronicidade da catarata hipermadura sugere-se que o comprometimento da estrutura capsular seja de intensidade equivalente e, por consequência, que isto também possa prejudicar o metabolismo das células do epitélio anterior da lente, diminuindo assim a incidência da opacidade da cápsula posterior de cães com catarata diabética e hipermadura submetidos à facoemulsificação.(AU)
Nowadays, the anterior lens capsule and its epithelium have been being frequently studied aiming to reduce the incidence of posterior lens capsule opacity, a complication that frequently occurs after surgical removal of cataracts, due to epithelium cells differentiation and migration to the posterior pole. The objective of this study was to evaluate by histochemistry (PAS and picrosirius red) analysis two important molecular components of the anterior lens capsule (proteoglycans and type IV collagen) in older diabetic and non-diabetic dogs, with diabetic and hypermature cataracts, after phacoemulsification surgery utilizing 0.1% trypan blue or not. Twenty seven dogs, including 21 female and 6 male dogs, with ages varying from 8 to 12 years old (mean = 9.6 yo) of different breeds were studied. The animals were divided into 2 groups: GC (hypermature cataracts) and GCD (diabetic cataracts). Results showed that, besides their different pathophysiologies, both types of capsules studied (diabetic and hypermature ones) presented the same molecular composition of proteoglycans and type IV collagen, since no statistical significant differences were observed. In addition, 0.1% trypan blue was not capable to induce any other evident alteration for the samples. In conclusion, our findings suggest that, if the results consist in alteration induced by the aggressive metabolism of the diabetic cataract or the chronicity of the hypermature one, it is of the same intensity and independent of the use of 0.1% trypan blue. It is also possible to suggest that this alteration must be capable to compromise lens epithelium cell metabolism, which should probably favour future lens posterior capsule studies.(AU)
Asunto(s)
Animales , Perros , Cápsula Anterior del Cristalino/patología , Catarata/complicaciones , Catarata/veterinaria , Diabetes Mellitus/veterinaria , Cápsula Posterior del Cristalino/cirugía , Proteoglicanos/análisis , Colágeno Tipo IV/análisis , Azul de Tripano , Facoemulsificación/veterinariaRESUMEN
Atualmente, a cápsula anterior e o epitélio da lente tem sido cada vez mais estudados, com o intuito de reduzir as possíveis complicações do pós-operatório da remoção da catarata, tal como a opacidade da cápsula posterior, alteração ocasionada principalmente pela diferenciação e migração das células do epitélio lenticular para a cápsula posterior da lente. O objetivo deste estudo foi analisar a composição molecular da cápsula anterior da lente pela técnica histoquímica de PAS (avaliação de proteoglicanos) e picrosirius red (avaliação de colágeno IV), em cães idosos com catarata diabética e não diabética do tipo hipermadura, submetidos ao uso ou não de azul de tripano a 0,1 % durante a facoemulsificação. Vinte e sete cães foram estudados, incluindo 21 fêmeas e 6 machos, de 8 a 12 anos de idade (média = 9,6 anos), de diversas raças e divididos em 2 grupos: GC (catarata hipermadura) e GCD (catarata diabética). Os resultados das análises realizadas mostraram que ambas as amostras, tanto as provenientes das cataratas hipermaduras, quanto das diabéticas, apresentam semelhante composição molecular de proteoglicanos e colágeno IV e isto independente da utilização de azul de tripano a 0,1 %. Conclui-se, portanto, que se os resultados obtidos forem decorrentes de alterações provocadas pelo rápido metabolismo da catarata diabética e pela cronicidade da catarata hipermadura sugere-se que o comprometimento da estrutura capsular seja de intensidade equivalente e, por consequência, que isto também possa prejudicar o metabolismo das células do epitélio anterior da lente, diminuindo assim a incidência da opacidade da cápsula posterior de cães com catarata diabética e hipermadura submetidos à facoemulsificação.
Nowadays, the anterior lens capsule and its epithelium have been being frequently studied aiming to reduce the incidence of posterior lens capsule opacity, a complication that frequently occurs after surgical removal of cataracts, due to epithelium cells differentiation and migration to the posterior pole. The objective of this study was to evaluate by histochemistry (PAS and picrosirius red) analysis two important molecular components of the anterior lens capsule (proteoglycans and type IV collagen) in older diabetic and non-diabetic dogs, with diabetic and hypermature cataracts, after phacoemulsification surgery utilizing 0.1% trypan blue or not. Twenty seven dogs, including 21 female and 6 male dogs, with ages varying from 8 to 12 years old (mean = 9.6 yo) of different breeds were studied. The animals were divided into 2 groups: GC (hypermature cataracts) and GCD (diabetic cataracts). Results showed that, besides their different pathophysiologies, both types of capsules studied (diabetic and hypermature ones) presented the same molecular composition of proteoglycans and type IV collagen, since no statistical significant differences were observed. In addition, 0.1% trypan blue was not capable to induce any other evident alteration for the samples. In conclusion, our findings suggest that, if the results consist in alteration induced by the aggressive metabolism of the diabetic cataract or the chronicity of the hypermature one, it is of the same intensity and independent of the use of 0.1% trypan blue. It is also possible to suggest that this alteration must be capable to compromise lens epithelium cell metabolism, which should probably favour future lens posterior capsule studies.
Asunto(s)
Animales , Perros , Catarata/complicaciones , Catarata/veterinaria , Cápsula Anterior del Cristalino/patología , Cápsula Posterior del Cristalino/cirugía , Diabetes Mellitus/veterinaria , Azul de Tripano , Colágeno Tipo IV/análisis , Facoemulsificación/veterinaria , Proteoglicanos/análisisRESUMEN
Cryopreserved peripheral blood mononuclear cells (PBMCs) are widely used in studies of dengue. In this disease, elevated frequency of apoptotic PBMCs has been described, and molecules such as soluble tumor necrosis factor (TNF)-related apoptosis-inducing ligands (sTRAIL) are involved. This effect of dengue may affect the efficiency of PBMC cryopreservation. Here, we evaluate the viability (trypan blue dye exclusion and amine-reactive dye staining) and functionality (frequency of gamma interferon [IFN-γ]-producing T cells after polyclonal stimulation) of fresh and cryopreserved PBMCs from children with dengue (in acute and convalescence phases), children with other febrile illnesses, and healthy children as controls. Plasma sTRAIL levels were also evaluated. The frequencies of nonviable PBMCs detected by the two viability assays were positively correlated (r = 0.74; P < 0.0001). Cryopreservation particularly affected the PBMCs of children with dengue, who had a higher frequency of nonviable cells than healthy children and children with other febrile illnesses (P ≤ 0.02), and PBMC viability levels were restored in the convalescent phase. In the acute phase, an increased frequency of CD3+ CD8+ amine-positive cells was found before cryopreservation (P = 0.01). Except for B cells in the acute phase, cryopreservation usually did not affect the relative frequencies of viable PBMC subpopulations. Dengue infection reduced the frequency of IFN-γ-producing CD3+ cells after stimulation compared with healthy controls and convalescent-phase patients (P ≤ 0.003), and plasma sTRAIL correlated with this decreased frequency in dengue (rho = -0.56; P = 0.01). Natural dengue infection in children can affect the viability and functionality of cryopreserved PBMCs.