Influência da diminuição da temperatura sobre o fuso meiótico de oócitos de camundongas e de mulheres maturados in vitro / Low temperature influence on meiotic oocyte spindle of mice and humans after maturation in vitro

Introdução: O fuso meiótico dos oócitos de mamíferos pode se despolimerizar quando exposto a pequenas variações de temperatura. Este fato já está bem estabelecido e estudado em oócitos maduros em metáfase II (MII). No entanto, pouco se sabe a respeito da influência da diminuição da temperatura sobre o fuso meiótico dos oócitos imaturos. Desse modo, este estudo tem como objetivos: 1) avaliar a influência da diminuição da temperatura sobre o fuso meiótico de oócitos de camundongas maturados in vitro e 2) avaliar o fuso meiótico em oócitos humanos maturados in vitro submetidos à criopreservação pela técnica de congelação lenta ou por vitrificação quando em estágio de vesícula germinativa. Métodos: Realizaram-se dois experimentos, denominados 1 e 2, sendo o primeiro em oócitos de camundongas e o segundo em oócitos humanos. No experimento 1 oócitos imaturos de camundongas nos estágios de metáfase I (MI), telófase I(TI) e MII foram cultivados nas seguintes temperaturas: 37º C (controle), temperatura ambiente (22oC) e 4º C por 0, 10, 30 e 60 minutos. Após este período de tempo o fuso meiótico oocitário foi avaliado por meio de microscopia de luz polarizada (MLP) (LC-Polscope-Oosight image software) e imunocitoquímica (IC). No experimento 2 oócitos em estágio de vesícula germinativa (GV) coletados de pacientes submetidas à indução da ovulação e fertilização in vitro, foram divididos de forma randômica em três grupos: oócitos a fresco (A), oócitos congelados pela técnica de congelação lenta (B) e oócitos congelados pela técnica de vitrificação (C). Os oócitos a fresco, os descongelados e os aquecidos foram maturados in vitro até estágio de (MII). A análise do fuso meiótico foi realizada por microscópio invertido equipado com uma câmera de vídeo analógica e um sistema de imagens que combina luz polarizada em cristal líquido (ICSI Guard Octax)...

Introduction: The meiotic spindle of most mammals is sensitive to cooling and depolymerizes even after a slight reduction in temperature. This is well described and studied on matured oocytes at metaphase II (MII). However, little is known about the influence of low temperatures under meiotic spindle of imature oocytes. In this way, we sougth to evaluate: 1) the influence of low temperatures on mice oocyte meiotic spindle matured in vitro e 2) the oocyte meiotic spindle from human oocytes matured in vitro and cryopreserved by slow-rate freezing or vitrification at GV stage. Methods: Two experiments were done: the first one on mice and the second one on women.At experiment 1, immature mice oocytes at metaphase I (MI), telophase I (TI) and MII were cultured at 37º C (control), room temperature (22oC) and 4º C for 0, 10, 30 and 60 minutes and then spindle analysis was made with polarized light microscopy (PLM) (LC-Polscope-Oosight image software) or immunocytochemistry (ICC). At experiment 2, GV oocytes retrieved from women submitted to ovulation induction and in vitro fertilization were randomly divided in three groups: fresh oocytes (A), cryopreserved by slow-freezing (B) and cryopreserved by vitrification (C). Fresh, thawed and warmed oocytes were matured in vitro to metaphase II oocytes (MII). A meiotic spindle analysis was done by polarized light microscopy (ICSI Guard Octax). Results: Experiment 1: At time 0 min and 37º C, all oocytes had polymerized spindles both at PLM or ICC. At 4º C, the number of MI oocytes with detectable spindles at PLM was smaller than those analysed by ICC, and it decreased with time, which had also occured with TI oocytes at a smaller proportion. However, at 4º C, TI meiotic spindle recognition with polarized light microscopy and ICC was comparable...

Análisis morfométrico del proceso de diferenciación in vitro de neuronas del hipocampo / Morphometric analysis of the differentiation process of hippocampal neurons in vitro

Los cultivos neuronales del sistema nervioso central se han venido usando ampliamente para el estudio de los mecanismos que conducen el proceso de diferenciación neuronal, así como también se han empleado como modelos in vitro para evaluar drogas y desarrollar nuevas terapias, de allí la importancia profundizar en la caracterización de dicho proceso. En este estudio, se prepararon cultivos primarios de células del hipocampo para estudiar los tipos celulares desarrollados, el desarrollo de dendritas y axones, la densidad de vesículas sinápticas y el desarrollo de los conos de crecimiento. Mediante inmunofluorescencia usando anticuerpos y marcadores no inmunológicos, se observaron los cambios experimentados por las estructuras de interés durante diferentes estadios temporales (1-21 días). Observamos una mayor proporción de neuronas sobre glias, desarrollo normal de las redes neuronales (conformadas por dendritas y axones), incremento en la longitud de dendritas y el establecimiento de sinapsis. Las vesículas sinápticas también experimentaron un incremento en su densidad a medida que aumentaba el tiempo de cultivo. Finalmente, se estudiaron los cambios morfológicos de los conos de crecimiento observándose que al inicio del cultivo en su mayoría se encontraban cerrados, pero a medida que maduraban las neuronas la proporción de conos de crecimiento abiertos aumentó. Este trabajo representa un avance en la caracterización morfométrica de los cultivos neuronales puesto que recoge de manera simultánea y cuantitativa los principales aspectos que marcan el proceso de diferenciación neuronal. En este estudio, la medición de estas características morfológicas hizo posible establecer parámetros cuantitativos que ayudarán a distinguir las principales etapas de la diferenciación neuronal.

Neuronal cultures of the central nervous system are widely used to study the molecular mechanisms that rule the differentiation process. These cultures have also been used to evaluate drugs and to develop new therapies. From this we can infer the relevance of performing an extended characterization that involves the main aspects driving such process. To carry out such characterization in the present study we prepared primary cultures from hippocampal cells to study cell identity, development of neuronal processes (dendrites and axons), density of synaptic vesicles and development of growth cones. Using immunofluorescence techniques, specific antibodies and non-immunological probes, we studied the changes experienced by the structures under study during different temporal stages (1-21 days). We observed a major proportion of neurons over glia, normal development of neuronal networks (formed by dendrites and axons), increase in the length of dendrites and axons and establishment of synaptic connections. Synaptic vesicles also showed an increase in their densities as long as the time of the culture progressed. Finally, we studied the morphological changes of the growth cones and observed that those were mostly closed at the beginning of the culture period. As neurons matured we observed an increase in the proportion of open growth cones. This work represents an advance in the morphometric characterization of neuronal cultures, since it gathers the main aspects that outline the neuronal differentiation process. In this study, measurement of these morphological features made possible to establish quantitative markers that will allow establishing more precisely the different stages of neuronal differentiation.

Efectividad del cálculo del poder dióptrico de la lente intraocular con interferometría parcialmente coherente / Effectiveness of the intraocular lens power calculation using the partial coherent interferometry

OBJETIVO: Comparar la efectividad del cálculo del poder dióptrico de la lente intraocular con IOL Master y el método de biometría por aplanación convencional. MÉTODOS: Se seleccionó una muestra de 100 ojos (pacientes) mediante un muestreo simple aleatorio, en el Servicio de Catarata del Centro de Microcirugía Ocular, con diagnóstico de catarata unilateral o bilateral en la consulta preoperatorio, desde marzo hasta septiembre de 2006. Se clasificaron en dos grupos según el método utilizado para el cálculo de la lente intraocular. Se analizaron las variables: longitud axial media preoperatoria, promedio queratométrico preoperatorio, componente esférico esperado y obtenido, agudeza visual sin corrección y mejor agudeza visual corregida preoperatoria y posoperatoria. El análisis estadístico de los resultados se realizó mediante un análisis de varianza, la prueba t de Student de comparación de medias para datos pareados y chi cuadrado. Se utilizó un nivel de confiabilidad de 95 por ciento. RESULTADOS: Entre los principales resultados se encontró que la diferencia de las longitudes axiales entre los métodos IOL Master y biometría por aplanación A-Scan fue estadísticamente significativa. La agudeza visual sin corrección aumentó cuatro líneas y la mejor agudeza visual corregida seis líneas en el posoperatorio de los pacientes del grupo I. El 90 por ciento de los pacientes del grupo I, o sea, los calculados con IOL Master quedaron en la emetropía en cuanto al componente esférico. CONCLUSIONES: Se evidenció una diferencia significativa e inferior a la encontrada en estudios internacionales entre las longitudes axiales preoperatorias halladas mediante los métodos IOL Master y biometría por aplanación; resultaron superiores las calculadas por IOL Master. Se obtuvo ganancia en las líneas de la Cartilla de Snellen tanto de la agudeza visual sin corrección como la mejor agudeza visual corregida en ambos grupos (superior en el grupo II). Predomina ron los resultados refrac...

OBJECTIVE: to compare the effectiveness of the intraocular lens dioptric power calculation using IOL Master and the conventional applanation biometry. METHODS: A sample of 100 eyes (patients), diagnosed with unilateral or bilateral cataract in the preoperative consultation service, was selected through simple random sampling in the Ocular Microsurgery Center in the period from March to September, 2006. They were divided into two groups based on the method for intraocular lens calculation. The variables were preoperative mean axial length, preoperative keratometric average, expected and obtained spheral component, visual acuity without correction and better corrected visual acuity preoperatively and postoperatively. The statistical analysis of the results was made by variance analysis, Student's t test for paired mean comparisons and Chi square. The confidence level of 95 percent was used. RESULTS: Among the main results, it was found that the axial length differences between IOL Master and A-Scan applanation biometry was statistically significant. Visual acuity without correction increased 4 lines and the best corrected visual acuity increased 6 lines in the postoperative period of the group I patients. Ninety percent of the group I patients, whose visual acuity was calculated with IOL Master, reached emetropia in terms of the spheral component. CONCLUSIONS: A significant difference but lower than that found in the international studies among the preoperative axial lengths calculated through ILO Master and applanation biometry were evinced. The differences were higher in the lenghts calculated by IOL Master. There was improvement in the number of lines of Snellen´s chart both in the visual acuity without correction and the better corrected visual acuity in the two groups; being better in group II. The refractive results tending to emetropy prevailed, taking into consideration the spheral component reached in both groups, which were also higher in group I

Proacrosin-acrosin activity in capacitated and acrosome reacted sperm from cryopreserved bovine semen

Acrosin activity is associated with normal fertility in human and bovine spermatozoa. The aim of the study was to determine the variation of the enzyme activity in the proacrosin-acrosin system in capacitated and acrosome recated cryopreserved bovine sperm. Enzyme activity was assessed spectrophotometrically using N-alpha-benzoyl-DL-arginine p-nitroanilide (BAPNA) as specific substrate for acrosin at pH 8. Capacitation with heparin and quercitin failed to induce conversion of proacrosin to acrosin. An increase in acrosin activity produced by the presence of progesterone, in a dose-dependent manner, was related with the induction of true acrosome reaction. The total level of acrosin activity registered showed that 96 per cent of acrosin of capacitated sperm samples and control is present in the zymogen form. Moreover, progesterone is capable of duplicating the level of active enzyme, indicating that enzyme activity changes are related to acrosome reaction, suggesting that only a minor proportion of the total of proacrosin-acrosin system is required in the exocytotic process on cryopreserved bovine sperm.

Ischiorectal endometria - a rare ischiorectal lesion

A female patient with 33 ages suffered the ischiorectal lesion that experienced incision without removal of lesion. The features suggested the ischiorectal endometria included solid organ caused pain in the ischiorectal region, no fever, increased pain and bigger organ during menstruation. The total removal operation of this organ and microscopic tests was carried out to treat and affirm the ischiorectal endometria

Morphological investigation of Toxoplasma gondii in vivo by a multiple beam interference microscope

A recently developed technique, namely multiple beam interference microscopy, has been applied to investigate the morphology of the parasite Toxoplasma gondii for the first time. The interference pattern obtained from the multiple internal reflection of a T. gondii, sandwiched between a glass plate and a cover plate, was focused on the objective of a conventional microscope. Because of the enhance contrast, several details of sub cellular structure and separating compartments are clearly visible. Details reveal the presence of a nucleus, lipid body, dense granule, rhoptry and amylopectin. The wall thickness of the membrane of the lipid body and the amylopectin is of the order of 0.02 æm and can be clearly distinguished with the help of the present technique. The same parasite has also been examined with the help of atomic force microscopy, and because of its thick membrane, the inner structural details were not observed at all. Sub cellular details of T. gondii observed with the present technique have been reported earlier only by low amplification transmission electron microscopy and not by any optical microscopic technique

Endoscopic appearance, dissecting microscopy and histopathology in malabsorption disorders

Small bowel biopsy is essential for the diagnosis of malabsorption and recently endoscopic inspection of duodenal mucosa was reported to be helpful in diagnosing cocliac disease. In a study of 214 patients suspected to have malabsorption, we compared the diagnostic yield of endoscopic appearance, dissecting microscopy and histopathology of small bowel biopsies. The endoscopic appearance was abnormal in 64%, 20.3%, 66.6% and dissecting microscopy showed abnormalities in 68.5%, 26.5% and 83.3% in celiac disease, tropical sprue and primary intestinal lymphoma respectively, reflecting the site of lesions in the small bowed in these disorders. Moreover, in organic causes of malabsorption the endoscopic appearance and dissecting microscopy showed a predictive value of 100% and 94.3% respectively. There was no statistical difference between both procedures in all the groups studied. It seems that endoscopic inspection of the duodenal mucosa and dissecting microscopy examination of the biopsy is safe, rapid, easy and reliable procedure in the diagnosis and differentiation of various syndromes of malabsorption

Una mirada retrospectiva sobre la evolucion historica de las formas del microscopio / Historic evolution of the microscopes

Se revisa, en forma somera, la evolicon que ha sufrido el microscopio de acuerdo a las diferentes epocas hasta llegar a la ultima creacion del MET y MEC.