Uso de Hidróxido de Sodio como Alternativa en Técnica de Diafanización de Diente / Use of Sodium Hydroxide as an Alternative in Tooth Clearing Technique

Con el objetivo de conservar material cadavérico, se han creado diferentes técnicas y/o soluciones donde una técnica es la diafanización dental para estudiar la morfología interna del diente. Esta técnica consta en trasparentar el tejido calcificado del diente haciendo visible los conductos radiculares al inyectar una mezcla colorante en ellos. Se han descrito diferentes variantes de la técnica de diafanización como la técnica de Okumura y la técnica de Robertson, pero ambas técnicas utilizan reactivos tóxicos o de difícil acceso, por lo que se ha realizado una búsqueda de reactivos de bajo costo y fácil acceso para realizar la técnica de diafanización, reportándose que la técnica de diafanización por maceración con KOH es válida para diafanizar dientes. El objetivo del presente estudio fue utilizar NaOH en la técnica de diafanización dental por maceración, como una variante de KOH al ser una base de similar característica que el KOH. Se utilizaron 13 dientes (siete tercer molares, cinco premolares y un canino) para realizar tres variantes de la técnica de diafanización: técnica de Robertson, por maceración con KOH y por maceración con NaOH utilizando barra agitadora y agitador magnético en los dientes. Con la técnica de Robertson se obtuvo un diente completamente transparentado, mientras que los dientes diafanizados por maceración, tanto con KOH y NaOH, se transparentaron menos, aunque se hicieron visibles los conductos radiculares, por lo que el uso de NaOH en la técnica de diafanización por maceración es válida, aunque requiere más tiempo que la maceración por KOH.

SUMMARY: To preserve cadaveric material, different techniques, and solutions have been created where one technique is dental diaphanization to study the internal morphology of the tooth. This technique consists of making the calcified tooth tissue transparent and making the root canals visible by injecting a dye mixture into them. Different variants of the diaphanization technique have been described, such as the Okumura and the Robertson techniques. However, both techniques use toxic or difficult-to-access reagents, so a search has been made for low- cost and easily accessible reagents to perform the diaphanization technique, reporting that the diaphanization technique by maceration with KOH is valid for the diaphanization of teeth. This study aimed to use NaOH in the dental clearing technique by maceration as a variant of KOH since it is a base with similar characteristics to KOH. Thirteen teeth (seven third molars, five premolars, and one canine) were used to perform three variants of the diaphanization technique: Robertson technique, KOH maceration, and NaOH maceration using a stirring bar and magnetic stirrer on the teeth. With the Robertson technique, a completely transparent tooth was obtained, while the teeth cleared by maceration, with both KOH and NaOH, were less transparent, although the root canals became visible. Therefore, using NaOH in the diaphanization technique by maceration is valid, although it requires more time than KOH maceration.

Assessment of multiple biomarkers in Lithobates catesbeianus (Anura: Ranidae) tadpoles exposed to zinc oxide nanoparticles and zinc chloride: integrating morphological and behavioral approaches to ecotoxicology.

The ecotoxicological risk to vertebrates posed by zinc oxide nanoparticles (ZnO NPs) is still poorly understood, especially in animals with a biphasic life cycle, which have aquatic and terrestrial phases, such as amphibians. In the present study, we investigated whether acute exposure (7 days) to ZnO NPs and zinc chloride (ZnCl2) at three environmentally relevant concentrations (0.1, 1.0, and 10 mg L-1) induces changes in the morphology, chondrocranium, and behavior of the tadpoles of Lithobates catesbeianus (Anura: Ranidae). Tadpoles exposed to both forms of Zn did not undergo any morphological or behavioral changes at the lowest concentrations (0.1 and 1.0 mg L-1). However, the animals exposed to the highest concentration (10 mg L-1) lacked oral disc structures, were smaller in size, had a longer tail, and presented changes in the position and coiling of the intestine and malformations of the chondrocranium in comparison with the control group. This indicates that ZnO NPs and ZnCl2 altered the development of the tadpoles, causing delays in their metamorphosis and even reducing individual fitness. The tadpoles exposed to both forms of Zn at 10 mg L-1 also had reduced mobility, especially in the presence of conspecifics. Based on these findings, we emphasize the importance of studying morphological, skeletal, and behavioral biomarkers to evaluate the toxic effects of metal-based nanoparticles in amphibians.

Histological analysis of an alkaline based tissue clearing technique for fetal musculoskeletal tissue / Análisis histológico de una técnica de limpieza de tejido de base alcalina para tejido musculoesquelético fetal

SUMMARY: Tissue clearing techniques are frequently used in the observation and description of anatomical structures and pathways without altering the three-dimensional layout of the anatomical specimen. Tissue optical clearing promotes preservation of three-dimensional structures, which allows the study of the internal anatomy in its original position and original spatial interaction. Among these techniques, Potassium Hydroxide (KOH) maceration clearing is one of the most widely used. However, the histological changes of tissue after KOH maceration have yet to be fully understood. Our aim is to describe the microscopical differences between macerated and normal tissue. To better understand said changes, two human fetuses with a gestation period of 16 to 28 weeks were cleared and processed for histological analysis. Microtome slides of the fetuses' lower limbs were obtained and stained with Hematoxylin & Eosin, Periodic Acid Schiff (PAS), and Masson's trichrome with the purpose of observing the histological and macromolecule composition changes in cleared tissue. Remarkable differences at a histological level regarding the composition of the cellular structures, since diaphanized tissues showed a predominance of extracellular matrix composed of collagen fibers with the absence of most of the nucleated cellular tissue. Phospholipid's saponification, nucleic acids degradation and a change on proteins structural properties are the main factors inducing clearing. At the same time, molecular stability of collagen in alkaline conditions allows the specimen to maintain its shape after the process.

RESUMEN: Las técnicas de limpieza de tejido se utilizan con frecuencia en la observación y descripción de estructuras y vías anatómicas sin alterar el diseño tridimensional de la muestra anatómica. El aclaramiento óptico de tejidos promueve la preservación de estructuras tridimensionales, lo que permite el estudio de la anatomía interna en su posición original y la interacción espacial original. Entre estas técnicas, el aclarado por maceración con Hidróxido de Potasio (KOH) es una de las más utilizadas. Sin embargo, los cambios histológicos del tejido después de la maceración con KOH aún no se han entendido por completo. Nuestro objetivo es describir las diferencias microscópicas entre el tejido macerado y el normal. Para entender mejor dichos cambios, dos fetos humanos con un período de gestación de 16 a 28 semanas fueron aclarados y procesados para análisis histológicos. Se obtuvieron microtomos de las extremidades inferiores de los fetos y se tiñeron con hematoxilina y eosina, ácido peryódico de Schiff (PAS) y tricrómico de Masson con el fin de observar los cambios histológicos y de composición de macromoléculas en el tejido aclarado. Diferencias notables a nivel histológico en cuanto a la composición de las estructuras celulares, ya que los tejidos diafanizados mostraban un predominio de matriz extracelular compuesta por fibras de colágeno con ausencia de la mayor parte del tejido celular nucleado. La saponificación de los fosfolípidos, la degradación de los ácidos nucleicos y un cambio en las propiedades estructurales de las proteínas son los principales factores que inducen la depuración. Al mismo tiempo, la estabilidad molecular del colágeno en condiciones alcalinas permite que la muestra mantenga su forma después del proceso.

Factors affecting secondary sex characteristics in the yellowtail tetra, Astyanax altiparanae.

This study interrogated factors which affect the appearance of secondary sexual characteristics, namely, fin spinelets (rigid dimorphic structure empirically associated with male sexual maturity in characids), in Astyanax altiparanae. Many variables such as the season of the year and several biotic components, including organism length, sex, phase of maturation, and the presence of gonads, were investigated. These factors were then associated with the physiological development of fin spinelets. The development of this trait is related to reproductive strategies but demonstrates considerable population variability as it is found throughout the year in some species but only during specific periods in others. Seventy-five specimens obtained from spontaneous spawn of farmed fish were arbitrarily grouped into small-, medium-, and large-sized groups in both summer and winter. Gonadal histology was performed to confirm each animal's sex and phase of maturation. Diaphanization of the fish was performed to visualize, count, and measure the fin spinelets. Finally, gonadectomization of some males was utilized to investigate the gonadal effect on the presence of fin spinelets. The present results show that the presence of fin spinelets is a secondary sexual characteristic of males which occurs independently of the season and is always present in males longer than 48 mm. However, in the summer, male specimens presented more rays with fin spinelets than during the winter. Furthermore, since fin spinelets were observed on immature males as well as spawning capable males, their presence cannot be directly associated with sexual maturity in male A. altiparanae, as previously supposed. Finally, gonadectomization resulted in an initial reduction in the length of fin spinelets. However, this trend was eventually normalized with time.

A novel, rapid technique for clearing leaf tissues.

PREMISE: Clearing leaves is a highly useful practice for many taxonomic, ecological, physiological, and eco-physiological aspects of research. Using traditional methods, the procedure for clearing a leaf (referred to as diaphanization) can take several days or even weeks. In our laboratory we developed a technique, originally used for dissociating wood, that yields excellent epidermal and leaf venation preparations clearly showing the details of epidermal cells, hydathodes, trichomes, leaf margins, and leaf venation, in a maximum of three days. METHODS AND RESULTS: A solution originally used to macerate wood (Franklin's solution) was used to accelerate the process of clearing leaves. Using this approach, it is possible to obtain clear images of the leaf adaxial and abaxial epidermal surfaces and leaf venation. Our technique works with both fragile and sturdy leaves, as well as with thin roots and stems. A few examples are presented. CONCLUSIONS: Franklin's solution is very simple to prepare and easy to manipulate. This new technique has the advantage of separating the leaf epidermal layers and venations, which can then be clearly observed.

Leaf architecture of Rubiaceae Juss. from caatinga vegetation in Brazil / Arquitetura foliar de espécies de Rubiaceae Juss. da caatinga brasileira

Abstract: The study and characterization of leaf venation plays a key role in the recognition of taxonomic groups that have been identified mainly based on reproductive traits. This study aimed at characterizing the pattern of leaf venation of Rubiaceae, recognizing individual characters among the taxa of this group and testing the use of leaf architecture as a taxonomic tool capable of providing vegetative and diagnostically characteristics between species. Leaves of 14 species distributed in eight genera from Rubiaceae were diaphanized and classified. The study area is located in Área de Proteção Ambiental Serra Branca/Raso da Catarina, Bahia State, Brazil. The characterization of secondary veins, including type, spacing and number of pairs, third veining patterns, fourth and fifth order, and the conformation of the areola were useful to delimit taxa when used together, providing subsidies to more comprehensive studies.

Resumo: O estudo e a caracterização da venação foliar representam uma possibilidade no reconhecimento de grupos taxonômicos, identificados predominantemente com base em caracteres reprodutivos. O presente estudo objetivou caracterizar o padrão de nervação foliar de Rubiaceae, reconhecer caracteres particulares dentre os táxons deste grupo e testar a utilização da arquitetura foliar como ferramenta taxonômica capaz de fornecer características diagnósticas vegetativas entre as espécies. Foram diafanizadas e classificadas as folhas de 14 espécies distribuídas em 8 gêneros de Rubiaceae na APA Serra Branca/Raso da Catarina. A caracterização das nervuras secundárias, incluindo tipo, espaçamento e número de pares, padrões de nervuras de terceira, quarta e quinta ordem, bem como a conformação das aréolas foram úteis para delimitar os táxons quando utilizados em conjunto, fornecendo subsidío para estudos mais abrangentes.

The use of Proteinase K to access genitalia morphology, vouchering and DNA extraction in minute wasps

ABSTRACT Genitalia are rich source of characters in insect taxonomy. Usually, they are examined after dissection and cleaning with potassium hydroxide (KOH), procedure that may damage both genital morphological structures and intracellular molecular contents. Enzymatic procedure with Proteinase K has been used to clean muscle off the genitalia while extract DNA, but its damage to the genital structures has not been evaluated. Herein, we qualitatively compare the use of KOH and Proteinase K to prepare genital structures in minute insects (Hymenoptera: Bethylidae). We show that Proteinase K is better to preserve the genital structure and provides quality DNA.

Leaf morphology and venation of Psidium species from the Brazilian Savanna

ABSTRACT The Brazilian Savanna biome has the largest plant diversity among savannas worldwide and is the predominant biome in Goiás state, Brazil. Among plant species previously catalogued in the Brazilian Savanna, the Myrtaceae family has received attention, as these plants show great economic potential for its medicinal properties, food and ecological importance, highlighting in this context the Psidium genus. In order to contribute to the differentiation of problematic taxonomic groups, such as Myrtaceae, and to quality control of its plant material, morphological and venation leaf studies of four species of Psidium were performed. For this purpose, leaf samples of Psidium firmum O.Berg., P. myrsinites DC., P. laruotteanum Cambes., and P. guineense Sw. were collected from Goiás State University, Anápolis Air Base, and Serra de Caldas Novas State Park and submitted to classical techniques for morphological and leaf venation characterizations. The results showed that P. firmum presents brochidodromous secondary veins, marginal last venation of the fimbrial type, an abaxial surface with a grooved midrib, flat secondary veins on both sides, an apex obtuse to mucronate, and a rounded base. P. guineense presents trichomes on both surfaces, a grooved midrib on the adaxial surface and a prominent midrib on the abaxial surface, which distinguishes this species from all other Myrtaceae species examined in the present study. The species P. guineense and P. firmum presented a set of differential leaf characteristics among the others taxa of the genus, clearly separating these plants in the morphological identification key.

Diaphanization: a standardized protocol for non-fetal tissue preservation / Diafanización: un protocolo estandarizado para la preservación de tejido adulto

In response to the arising difficulty of dissection use during anatomy courses, medical schools have been forced to research alternative teaching methods. These are meant to help students develop three dimensional mental images of the human body and increase spatial reasoning, thus improving the learning process of human morphology. One of those methods, used at the Universidad de los Andes is the Diaphanization process. This technique is a well-known method for specimen preservation, used as an anatomy research and teaching tool. Even though it is frequently used, finding a standardized protocol in indexed journals is not possible, which hinders the use of the technique. The standardization of an updated protocol is a need in order to continue exploiting the maximum educational capacity of the specimens used during classwork. The process used at the Universidad de los Andes for non-fetal tissue preservation by Diaphanization is described, with a detailed explanation of its five main stages: Injection, Fixation, Dehydration, Bleaching (or Maceration) and Final preservation. The final result should be an organ preserved in an acrylic box which allows clear three dimensional visualization of anatomical structures that can be used for the study and description of general anatomy and vascular structure. It also helps with spatial reasoning and represents little to no biological risk, leading to a new level of anatomical teaching and experimenting. Several specimens obtained at our laboratory through this technique are presented.

Como respuesta a la dificultad del uso de disección como una herramienta en el curso de anatomía, las escuelas de medicina se han visto forzadas a investigar y desarrollar métodos alternativos que la reemplacen. Estos pretenden estimular el desarrollo de la percepción tridimensional de los estudiantes, logrando mejor racionamiento espacial, y así, mejorando el proceso de aprendizaje de la morfología humana. Uno de estos métodos, en uso en la Universidad de los Andes, es el proceso de diafanización. Esta técnica es ampliamente conocida en el ámbito de preservación de tejidos, y empleada en investigación y educación. Aún con su uso frecuente, un protocolo estandarizado que la describa no está disponible en la literatura indexada, lo cual limita su uso. La estandarización de un protocolo actualizado es necesaria para permitir el continuo uso de esta técnica. Se describe el proceso de preservación de tejido adulto mediante la diafanización, con una explicación detallada de los 5 pasos que la componen: Inyección, Fijación, Deshidratación, Macerado y Preservación final. El resultado es un órgano inmerso en glicerina en un contenedor de acrílico que permite una visualización tridimensional de las estructuras anatómicas del espécimen, que puede ser empleado por los estudiantes para su estudio, y permite una descripción de la anatomía general del modelo y su estructura vascular. Adicionalmente incrementa el razonamiento espacial de los estudiantes, y no presenta ningún riesgo biológico. Se presentan una variedad de especímenes de características particulares obtenidos mediante la aplicación de esta técnica.

Estudo da anatomia interna do canal radicular em incisivos inferiores pela técnica de diafanização / Root canal anatomy study of lower incisors by the clearing technique

Para o sucesso da terapia endodôntica, é indispensável o pleno conhecimento da anatomia radicular interna, assim como suas variações. Os incisivos inferiores são os menores dentes da arcada humana e apresentam uma raiz fortemente achatada no sentido mesodistal. Esse achatamento é, por vezes, tão grande que determina a divisão do canal radicular em dois, um vestibular e um lingual. O objetivo deste estudo foi analisar por intermédio de diafanização (método que permite observar por transparência a diversificada anatomia radicular) a incidência do número de canais presentes nos terços cervical, médio e apical da raiz de incisivos inferiores humanos. Para isso foram obtidos 150 dentes no banco de dentes da Faculdade de Odontologia da Universidade de Passo Fundo, nos quais se aplicou a técnica de diafanização e injeção de tinta nanquim no interior da câmara pulpar, seguida de conservação em salicilato de metila, o que permitiu observar tridimensionalmente, por transparência, a anatomia interna dos canais radiculares. Os resultados foram submetidos à análise estatística descritiva, e notou-se que 81,63% dos espécimes analisados apresentaram um canal no terço cervical, 53,06% tinham dois canais no terço médio e 71,42% possuíam um canal no terço apical. Com isso é possível concluir que há maior incidência de bifurcação do canal radicular no terço médio da raiz em relação aos terços cervical e apical

To succeed in the endodontic therapy, it's a requisite to have full knowledge of the inner root anatomy, as well as its variations. The lower incisors are the tinniest teeth of the human arcade having a root strongly flattened to the mesio-distal way. This flattening is sometimesas big to split the root canal in two, one vestibular and another lingual.This study has utilized the clearing technique (method that allows tosee through transparency the diverse root anatomy) to accomplish its objective of analyzing the incidence of the canals present in the cervical third, middle and apical of the incisors' roots of human lowers. Fort hat purpose, 150 teeth have been obtained from the Teeth Bank of the Odontology Faculty of the University of Passo Fundo, on which the clearing technique and the injection of Naquin ink in the pulp cavity interior were applied, followed by the conservation in the methylsalicylate, allowing to three-dimensionally observe, through transparency, the internal anatomy of the root canal. The results have been analyzed by descriptive statistics, in which it was observed that81,63% of the analyzed samples have presented one canal in the cervical third; 53,06% have two canals in the middle third and 71,42% have one canal in the apical third. With this it can be concluded that there is a major incidence of the root canal bifurcation in the middle third of the root in relation to the cervical and apical