An evaluation of an animal model of cerebral palsy: the effects on the morphology of the extensor digitorum longus muscle / Evaluación de un modelo animal de parálisis cerebral: los efectos sobre la morfología del músculo extensor largo de los dedos

Cerebral Palsy (CP) refers to chronic childhood encephalopathy. The objective of this study was to verify effects of CP model that combines prenatal exposure to LPS, perinatal anoxia and sensorimotor restriction on EDL muscle. Male Wistar rat pups were separated: a) Control - pups of mothers injected with saline during pregnancy and b) Cerebral Palsy - pups of mothers injected with LPS during pregnancy, and submitted to perinatal anoxia and sensorimotor restriction. The CP group presented hypertrophy in the type IIB fibers and increase of nuclei/fiber and capillary/fiber ratios. The intrafusal fibers of CP group presented 26 % atrophy in the crosssectional area and intramuscular collagen volume increase 34 %. CP group showed myofibrillar disruption and Z-line disorganization and the NMJs presented increases of 22 % in area.This animal model of CP produces motor deficits and macro and microscopic alterations and in the ultrastructure of the EDL muscle.

La parálisis cerebral (PC) se refiere a la encefalopatía crónica infantil. El objetivo de este estudio fue verificar los efectos del modelo PC que combina la exposición prenatal a LPS, la anoxia perinatal y la restricción sensitivo-motora en el músculo extensor largo de los dedos (MELD). Se separaron las crías de ratas Wistar machos: a) Control: crías de madres inyectadas con solución salina durante la preñez y b) Parálisis cerebral: crías de madres inyectadas con LPS durante la preñez y sometidas a anoxia perinatal y restricción sensitivo-motora. El grupo PC presentó hipertrofia en las fibras tipo IIB y aumento de la relación núcleo / fibra y capilar / fibra. Las fibras intrafusales del grupo PC presentaron un 26 % de atrofia en el área de la sección transversal y el volumen de colágeno intramuscular aumentó un 34 %. El grupo PC mostró disrupción miofibrilar y desorganización de la línea Z y los NMJ presentaron aumentos de 22 % en el área. Este modelo animal de PC produce déficit motores y alteraciones macro y microscópicas y cambios en la ultraestructura del MELD.

Morphological and morphometric study of the muscle fibers and neuromuscular junctions of the extensor digitorum longus in aged rats submitted to maternal protein restriction / Estudio morfológico y morfométrico de las fibras musculares y de las intersecciones neuromusculares del músculo extensor largo de los dedos de ratas de edad avanzada sometidas a la restricción proteica materna

Protein restriction implies the functional involvement of several systems and organs, including the skeletal muscle, because it is a protein reservoir in the body. This study sought to analyze the morphological and morphometric features of the muscle fibers and neuromuscular junctions (NMJs) of the extensor digitorum longus (EDL) muscle in rats at 365 days of age, submitted to maternal protein restriction during the gestation and lactation periods. Wistar rats were divided into two groups: a Control Group - mothers fed a normal-protein diet (17 % protein) during pregnancy and lactation; and a Restricted Group - mothers fed a low-protein diet (6 % protein) during pregnancy and lactation. The pups were kept with the mother throughout the lactation period (21 days), after which the offspring received a normal protein diet until 365 days of age. Histological (HE) and histoenzymological (NADH-TR) studies were conducted on the muscle fibers. The muscle was subjected to Nonspecific Esterase reaction to stain the Neuromuscular Junctions. Regarding the animals from the restricted group: the histologic analysis of the muscle fibers showed the presence of centralized nuclei and a diminished area; the histoenzymological study showed the different types of muscle fibers were randomly distributed in the EDL muscle and the area of the Type IIa muscle fiber was smaller; the ultrastructural study revealed disorganization of the Z line, and the presence of lipid droplets and vacuoles containing myelin figures in subsarcolemmal and intramiofibrilar regions; while the analysis of the NMJs exhibited no significant differences between the groups. Protein restriction in the pregnancy and lactation period may have affected the development of skeletal muscle, producing a permanent muscle-fiber deficit in the EDL muscle of the offspring.

La restricción proteica implica compromiso funcional de diversos sistemas y órganos, entre ellos, el músculo estriado esquelético, por ser una reserva de proteína del organismo. De esa forma, el presente trabajo procuró analizar las características morfológicas y morfométricas de las fibras musculares y de las intersecciones neuromusculares (JNMs) del músculo extensor largo de los dedos (EDL) en ratas de 365 días de edad, sometidas a restricción proteica materna durante los periodos de gestación y lactancia. Las ratas Wistar fueron separadas en dos grupos: El grupo Control - madres alimentadas durante la gestación y lactancia con ración normoproteica (17 % de proteína) y Grupo con restricción ­ madres alimentadas durante la gestación y lactancia con ración hipoproteica (6 % de proteína). Las crías permanecieron con la madre durante todo el periodo de lactancia (21 días) y después de este periodo la prole recibió ración normoproteica hasta los 365 días de edad. Se realizó un estudio histológico (HE) e histoenzimológico (NADH-TR) de las fibras musculares. Para la marcación de las JNMs, el músculo fue sometido a la reacción de Esterasa Inespecífica. El análisis histológico de las fibras musculares de los animales del Grupo con restricción mostró la presencia de núcleos centralizados y una disminución del área en el grupo con restricción. En el estudio histoenzimológico, el músculo EDL presentó una distribución aleatoria de los diferentes tipos de fibras musculares y el área de las fibras musculares del tipo IIa fue menor en el grupo con restricción. En relación al estudio ultraestructural, en los animales del grupo con restricción se observó desorganización de la línea Z, presencia de pequeñas gotas de lípidos y vacuolas que abrigaban figuras de mielina en las regiones subsarcolemal e intramiofibrilar. En el análisis de las JNMs no hubo diferencias significativas. La restricción proteica impuesta en el periodo de gestación y lactancia puede haber afectado el desarrollo del músculo esquelético, produciendo un déficit permanente en las fibras musculares del músculo EDL de la prole.

Effects of immobilization and remobilization on the ankle joint in Wistar rats

A sprained ankle is a common musculoskeletal sports injury and it is often treated by immobilization of the joint. Despite the beneficial effects of this therapeutic measure, the high prevalence of residual symptoms affects the quality of life, and remobilization of the joint can reverse this situation. The aim of this study was to analyze the effects of immobilization and remobilization on the ankle joint of Wistar rats. Eighteen male rats had their right hindlimb immobilized for 15 days, and were divided into the following groups: G1, immobilized; G2, remobilized freely for 14 days; and G3, remobilized by swimming and jumping in water for 14 days, performed on alternate days, with progression of time and a series of exercises. The contralateral limb was the control. After the experimental period, the ankle joints were processed for microscopic analysis. Histomorphometry did not show any significant differences between the control and immobilized/remobilized groups and members, in terms of number of chondrocytes and thickness of the articular cartilage of the tibia and talus. Morphological analysis of animals from G1 showed significant degenerative lesions in the talus, such as exposure of the subchondral bone, flocculation, and cracks between the anterior and mid-regions of the articular cartilage and the synovial membrane. Remobilization by therapeutic exercise in water led to recovery in the articular cartilage and synovial membrane of the ankle joint when compared with free remobilization, and it was shown to be an effective therapeutic measure in the recovery of the ankle joint.

Cells of the rectum of Bombyx mori affected by experimental inoculation with Alphabaculovirus.

Bombyx mori is an insect whose cocoon is used in the sericulture industry, which is an important activity in parts of southern Brazil. When parasitized by Bombyx mori nucleopolyhedrovirus (BmNPV) of the genus Alphabaculovirus (alphaBV), it develops nuclear polyhedrosis disease. In Brazil, an alphaBV was isolated from larvae of B. mori and various target tissues were identified. However, how this geographic viral isolate affects the rectum of silk moths was unknown. The rectum, a component of the cryptonephric system, acts to absorb water and mineral salts, and its importance for the metabolic balance of insects provoked interest in analyzing how it is affected by BmMNPV infection. Fifth instar B. mori larvae were inoculated with a viral suspension, and from the second to the ninth day post-inoculation, segments of the rectum (anterior and anal canal) were examined using light microscopy. The cryptonephric epithelial cells in the anterior region revealed no evidence of infection. However, from the fifth day post-inoculation, cells of the anal canal showed cytopathologies characteristic of alphaBV. Infection of the anal canal and other surrounding tissues led to tissue disorganization, with accumulation of polyhedra in the perinephric space and compartmentalization of the cryptonephric system, promoting changes in the fecal pellets, signalling physiological changes. These observations contribute to our understanding of the infectious cycle of BmMNPV in B. mori.

Effects of immobilization and remobilization on the ankle joint in Wistar rats.

A sprained ankle is a common musculoskeletal sports injury and it is often treated by immobilization of the joint. Despite the beneficial effects of this therapeutic measure, the high prevalence of residual symptoms affects the quality of life, and remobilization of the joint can reverse this situation. The aim of this study was to analyze the effects of immobilization and remobilization on the ankle joint of Wistar rats. Eighteen male rats had their right hindlimb immobilized for 15 days, and were divided into the following groups: G1, immobilized; G2, remobilized freely for 14 days; and G3, remobilized by swimming and jumping in water for 14 days, performed on alternate days, with progression of time and a series of exercises. The contralateral limb was the control. After the experimental period, the ankle joints were processed for microscopic analysis. Histomorphometry did not show any significant differences between the control and immobilized/remobilized groups and members, in terms of number of chondrocytes and thickness of the articular cartilage of the tibia and talus. Morphological analysis of animals from G1 showed significant degenerative lesions in the talus, such as exposure of the subchondral bone, flocculation, and cracks between the anterior and mid-regions of the articular cartilage and the synovial membrane. Remobilization by therapeutic exercise in water led to recovery in the articular cartilage and synovial membrane of the ankle joint when compared with free remobilization, and it was shown to be an effective therapeutic measure in the recovery of the ankle joint.

Bombyx mori pylorus infection by Alphabaculovirus.

Alphabaculovirus is an entomopathogenic virus genus that infects Bombyx mori, which is known as the Bombyx mori multiple nucleopolyedrovirus (BmMNPV). This virus is polyorganotrophic, and a series of tissues are known as targets; however, there is currently no information regarding infection in the pylorus, the segment of the hindgut that is present in the midgut transition and is responsible for food passage control. Thus, in the present study, we aimed to analyze infection of the B. mori pylorus by BmMNPV. To do so, hybrid B. mori larvae were inoculated with a viral suspension of BmMNPV, and segments of the intestine containing the pylorus and its subdivisions, the posterior interstitial ring (PIR), pyloric cone, and pyloric valve, were dissected and processed for light microscopy on different days post inoculation. The results showed that B. mori pylorus subdivisions respond differently to infection, and the anterior area of the PIR is susceptible with these cells being the secondary infection targets. Cytological analysis revealed the presence of viroplasm in the hypertrophic nucleus, followed by the formation and development of viral polyhedra. Cytolysis occurred at the end of the infectious cycle, thereby releasing polyhedra and enabling the spread of the disease. There was no evidence of BmMNPV infection in the posterior area of the PIR, cone, or pyloric valve. These results will contribute to greater understanding of the virus infectious cycle, whose consequent epizootic disease can negatively impact this economically important insect that is used in silk production in Brazil.

Morphology of the Pylorus of Bombyx mori (Linnaeus) (Lepidoptera: Bombycidae).

The pylorus is a segment of the hindgut of great morphological diversity, which is subdivided into the posterior interstitial ring, pyloric cone, and pyloric valve. There is no morphological information on Bombyx mori (L.) hybrids, which is an insect of economic interest to Brazil. Consequently, this study aimed to analyze the morphology of the pylorus of hybrid larvae of fifth instars of B. mori from crossbreeding of Chinese and Japanese races. These were anesthetized, and the pylorus was fixed and processed by light microscopy and scanning electron microscopy. The pylorus presented as a separate compartment, consisting of a simple epithelium, with large variation in cell morphology, covered on the luminal side by an intima with specializations in some areas, the spicules. The musculature was formed by bundles of striated muscle fibers in longitudinal and circular arrangement. These fibers were multinucleated with central and peripheral nuclei, operating in peristalsis. The morphology of the pylorus is a reflection of functional dynamism, and this study provided a better understanding of its physiology, which promotes the regulated movement of the bolus, preventing reflux and directing it towards the ileum in the formation of fecal pellets.

Virucidal evaluation of sodium dichloroisocyanurate formulation against nucleopolyhedroviruses of silkworm / Avaliação antiviral do formulado de sódio dicloroisocianurato contra o nucleopoliedrovírus do bicho-da-seda

This study evaluated the effectiveness of a disinfectant with low corrosive action and which is not toxic to the environment, the sodium dichloroisocyanurate formulation, on the Bombyx mori nucleopolyhedrovirus (BmNPV). For this, 5th-instar B. mori silkworm larvae were divided into four experimental groups of 4 replicates with 15 larvae each, totalling 60 larvae per group. The groups were fed with mulberry (Morus sp.) leaf discs containing: BmNPV treated with the disinfectant, untreated BmNPV, only the disinfectant, and water (control). The results showed that the disinfectant does not inactivate the BmNPV and also exerts a negative effect on the insect"s resistance.

O estudo avaliou a eficiência de um desinfetante que apresenta baixa ação corrosiva e que não é tóxico ao meio ambiente, o formulado de sódio dicloroisocianurato, sobre o nucleopoliedrovírus Bombyx mori (BmNPV). Para tanto, lagartas do bicho-da-seda, B. mori, de 5º instar foram divididas em quatro grupos experimentais, 4 repetições com 15 lagartas cada, totalizando 60 lagartas por grupo. Os grupos foram alimentados com discos foliares de amoreira (Morus sp.) contendo: o BmNPV tratado com o desinfetante (solução 1); o BmNPV não tratado (solução 2); apenas o desinfetante (solução 3); e água (solução 4, controle). Os resultados mostraram que o desinfetante não inativa o BmNPV e também exerce efeito negativo na resistência do inseto.

Silkworm salivary glands are not susceptible to Bombyx mori nuclear polyhedrosis virus.

A nuclear polyhedrosis virus isolated from infected Bombyx mori, BmNPV, was used to inoculate silkworms to determine salivary gland cell susceptibility. The salivary gland was removed from infected silkworms at different times post-inoculation and examined by light microscopy. The salivary gland cells did not exhibit any signs of BmNPV infection; however, fat body and tracheal cells, used as positive controls, showed characteristic cytopathological changes caused by BmNPV infection, which confirmed inoculum viability. The morphological distribution of tracheal branches and the basal lamina, which serves as a barrier to viral penetration, are apparently involved in this resistance to infection.

Molecular markers in commercial Bombyx mori (Lepidoptera: Bombycidae) hybrids susceptible to multiple nucleopolyhedrovirus.

The silkworm Bombyx mori L. is particularly susceptible to virus diseases, especially B. mori nucleopolyhedrovirus (BmNPV). Disease resistance, along with high productivity, are important selection criteria for developing commercial hybrids of B. mori. We used bioassays and molecular markers linked to susceptibility/resistance to baculovirus infection to analyze the response of commercial B. mori hybrids from two companies to a geographic isolate of B. mori multiple nucleopolyhedrovirus (BmMNPV) from Paraná state in Brazil. Both of these commercial lines were highly susceptible to BmMNPV, with death rates of 92 and 94%. A polymorphic fragment of approximately ~350 bp, associated with susceptibility, and an ~800-bp fragment, associated with resistance to BmMNPV, were detected in all specimens. An additional fragment of ~480 bp, recently identified by our research team as a BmMNPV genomic sequence, was detected in the infected silkworms and could be used as a molecular marker for the diagnosis of nucleopolyhedrovirus infection.

Cytopathology of Bombyx mori (Lepidoptera: Bombycidae) silk gland caused by multiple nucleopolyhedrovirus.

A multiple nucleopolyhedrovirus previously isolated from infected Bombyx mori L. larvae (BmMNPV) in Paraná state, Brazil, was inoculated into B. mori larvae to examine susceptibility and cytopathology in silk gland cells. The anterior, middle and posterior silk glands were removed from the infected silkworm at different times post-inoculation and processed for cytopathology studies by light and transmission electron microscopy. BmMNPV infection was only detected at 72 h post-inoculation in cells of the middle and posterior silk glands. No sign of infection was found in the anterior silk gland. Cytopathological characteristics were similar to those found in other target tissues; initially, they consisted of nuclear hypertrophy and the formation of virogenic stroma (viroplasm), in which the progeny virions are produced. The virions are then enveloped and occluded in protein crystal structures, the polyhedra. After viral replication, cells undergo lysis and release a great number of BmMNPV polyhedra into the hemocoele. Histopathology showed early infection foci in regions surrounding trachea insertions, demonstrating that trachea is an infection-spreading organ in the insect body. Trachea penetrates the middle and posterior silk gland basal lamina, considered a barrier to virus, facilitating the penetration of budded virus. The anterior silk gland does not have tracheal insertions into the basal lamina, which reduces budded virus infection. Tracheal branches provide a conduit for non-occluded virions (budded virus) to pass through the basal lamina barrier and disseminate BmMNPV in the silkworm silk gland.

Sistema automatizado para análise de cariótipos humanos / Automated system for human karyotyping

A análise de cariótipos humanos, procedimento padräo em investigaçöes genéticas, é uma técnica complexa que demanda tempo, custo elevado, aliada ao trabalho de especialistas para a realizaçäo de um diagnóstico seguro. Tendo em vista estas características foi desenvolvido um sistema automatizado para análise de cariótipos humanos, na linguagem Delphi 3.0 Client/Server Suite e em ambiente Microsoft Windows 98. O sistema captura imagens de cromossomos metafásicos, diretamente do microscópio acoplado a uma câmara filmadora. Estas imagens säo enviadas para uma unidade central de processamento, onde o software, organiza os pares cromossômicos pelo tamanho. Durante este procedimento uma série de rotinas permitem ao usuário näo só a manipulaçäo da imagem como um todo, mas também, dos cromossomos individualmente. O cariótipo, assim obtido, pode ainda ser comparado com um padräo de bandas existentes, permitindo os ajustes finais. O software agiliza a análise cariotípica, dispensando alguns procedimentos manuais como o registro fotográfico, o recorte e a montagem manual dos pares cromossômicos. Os custos da análise também säo reduzidos, tanto em termos materiais, quanto humanos, uma vez que se reduz o tempo gasto pela especialista nas análises

Estudo morfológico das granulaçöes aracnóides humanas com referência a sua classificaçäo / Morphological study of human arachnoid granulations regarding their classification

O estudo das granulaçöes aracnóides humanas por esteromicroscópio e microscópio óptico revelou que em um mesmo indivíduo estäo presentes granulaçöes morfologicamente distintas, que classificamos em simples e lobuladas. As granulaçöes simples eram pequenas e completamente envoltas por cápsula fibrosa que delimitava, em torno das granulaçöes, espaço subdural contínuo desdce o pedículo até o ápice. As granulaçöes lobuladas eram maiores que as simples; em seu ápice a cápsula era delgada e ocorria interrupçäo do espaço subdural, devido à fusäo do tecido fibroso da cápsula com a periferia da granulaçäo. As granulaçöes simples estavam possivelmente em fase inicial de desenvolvimento, enquanto as granulaçöes lobuladas estariam em fase mais avançada, com estrutura morfológica ideal para absorçäo do LCR