Viability of primordial follicles derived from cryopreserved ovine ovarian cortex tissue

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Detection of bovine Herpesvirus type 5 in formalin-fixed, paraffin-embedded bovine brain by PCR: a useful adjunct to conventional tissue-based diagnostic test of bovine encephalitis

The aim of this study was to evaluate the application of PCR technique for the detection of BoHV-5 in routinely formalin-fixed, paraffin-embedded brain tissues in 20 naturally infected calves affected by fatal meningoencephalitis. Brains were divided into two halves, one kept fresh for virus isolation and PCR assay, targeting the glycoprotein C gene from BoHV-5 genome. The other half brain, corresponding to posterior cortex region, was submitted to formalin fixation and embedded into paraffin blocks for microscopic evaluation and total DNA isolation. Most of the slides showed severe multifocal non-supurative encephalitis with neuronal degeneration, neurophagia, and no acidophilic intranuclear inclusions could be found in neurons and glial. The 20 fresh samples were confirmed, by virus isolation and PCR assay, as having the BoHV-5 virus and, respective glicoprotein C sequence, while 15 of 20 formalin-fixed, paraffin-embedded samples were considered positive for the same analysis. The results revealed the first description of PCR efficiency, applied to formalin-fixed, paraffin-embedded brain collected from naturally infected calves, improving the detection of BoHV-5 from archival samples in South America. (c) 2007 Published by Elsevier B.V.

Histopathology of the reproductive system of male sheep experimentally infected with Toxoplasma gondii

The aim of this study was to investigate the histopathological changes in reproductive system (testicles, epididymis, seminal vesicles, and prostate) of small male ruminants after Toxoplasma gondii infection. Eight sheep were inoculated with T. gondii: group I, four sheep (2.0 x 10(5) P-strain oocysts); group II, four sheep (1.0 x 10(6) RH-strain tachyzoites); and group III, two uninfected sheep maintained as control. Infection with T. gondii was confirmed by seroconversion (indirect fluorescent antibody test-IgG) in all the infected animals beginning on post-inoculation day (PID) 7. on PID 70, all the animals were euthanized and tissue samples (testicles, epididymis, seminal vesicles, and prostate) were collected and processed for histological analysis. The main changes detected were a focal mononuclear interstitial inflammatory infiltrate in the prostate and seminal vesicles; diffuse testicular degeneration associated with calcification foci and a multifocal mononuclear interstitial inflammatory infiltrate; and a mononuclear interstitial infiltrate and focal necrotic areas of the muscle fibers surrounding the seminal vesicles. The histopathological findings of this work, along with the detection of T. gondii in the examined parenchyma tissues (immunohistochemistry) and the results obtained by other authors examining different tissues, suggest that histological changes diagnosed in the reproductive system of rams infected with T. gondii are strongly suggestive of toxoplasmatic infection.

Light-Emitting Diode Therapy in Chemotherapy-Induced Mucositis

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Morphometric and morphological analysis of neuromuscular junction alterations in the denervated rat diaphragm

The morphological and structural alterations that occur in the neuromuscular junctions of the denervated rat diaphragm were studied. Fifteen adult male albino rats (Rattus norvegicus) aged about 60 days and with a mean weight of 200 g were used. Chronically denervated diaphragms were obtained and the animals were sacrificed after 4, 8 and 12 weeks of denervation. The left antimere of the diaphragm was denervated by section of the phrenic nerve and the right antimere was used as control. Each antimere was divided into three fragments: one was used for histochemical (nonspecific esterase) and morphometric study of neuromuscular junctions, and the other two were used for transmission and scanning electron microscopy (SEM) analysis. Histochemical analysis of the diaphragm neuromuscular junctions after denervation showed only small changes in junction morphology. However, these junctions became smaller and elongated and presented less visible contours with increasing time of denervation. Ultrastructural analysis of neuromuscular junctions after 12 weeks showed more or less organized junctional folds on the muscle fiber surface. The junctional cytoplasm exhibited important alterations such as mitochondrial degeneration and the presence of numerous filaments. SEM revealed the presence of deep primary synaptic grooves with peripheral excavations which housed the nerve terminal boutons and exhibited internally the secondary synaptic clefts present among the junctional folds of the sarcolemma. This study showed that some of the morphological changes demonstrated in other denervated striated skeletal muscles are not repeated at the same intensity or in the same temporal pattern in the rat diaphragm.

Ultrastructure and cytochemistry of the pearl gland in Piper regnellii (Piperaceae)

Pearl glands are scattered throughout the lamina of developing leaves and rarely found on adult leaves of Piper regnellii (Piperaceae). The pearl gland is a bicellular secretory trichome composed of a short broad basal cell and a spatula-like, semiglobular apical cell. Four different stages of the pearl grand were determined during its ontogenesis: origin, pre-secretory, secretory and post-secretory. During the pre-secretory stage, mitochondria, ribosomes, dictyosomes, rough endoplasmic reticulum, and plastids with electron dense inclusions were present in the cytoplasm of the apical cell. During the secretory stage, the most remarkable characteristics of the apical cell are the proliferation of dictyosomes and their vesicles, rough endoplasmic reticulum, and modified plastids. At this stage, electron-dense oil drops occur in the plastids as well as scattered within the cytoplasm, proteins and polysaccharides are seen in the plastids, vesicles, and vacuoles. Only polysaccharides are present in the periplasmic space, wall cavities, and on the surface of the apical cell. The polysaccharides are one of the main components of the mucilagenous exudate that covers the developing leaf structures. The apical cell of the senescing trichomes undergoes a progressive degeneration of its cellular components, the plastids being the first organelles to undergo lysis.

Structural and cytochemical changes in the salivary glands of the Rhipicephalus (Boophilus) microplus (CANESTRINI, 1887) (Acari : Ixodidae) tick female during feeding

This study describes the morphology of salivary glands of Rhipicephalus (Boophilus) microplus female ticks at beginning of feeding (24-48 h of attachment) and semi-engorged (4-5 days of attachment) to verify the degenerative characteristics of these organs and the secretory phase in which the process begins. At the beginning of feeding, secretion granules had been observed only in the cytoplasm of cells b, c(1), c(2), c(4) (type II acinus) and d (type III acinus), as well as large nuclei with regular and preserved morphology. In the semi-engorged females the acini presented few normal cells, few partially preserved ones, and the remaining ones in several stages of degeneration, that is, with retraction and cytoplasmic vacuolization, and nuclei with chromatin in several stages of condensation, picnotic and/or in fragmentation. In type I acinus and in the excretory ducts of the studied glands, at both feeding stages, no degenerative characteristic was observed. In females of R. (B.) microplus, the salivary glands degenerate asynchronically and precociously when compared with those of others tick's species. (c) 2006 Elsevier B.V. All rights reserved.

Ultrastructural modifications in the venom glands of workers of Apis mellifera L. (Hymenoptera : Apidae) promoted by topical application of juvenile hormone

The present study analyzed, the influence of the treatment with juvenile hormone on the ultrastructure of Apis mellifera L. workers' venom glands. Newly emerged workers received topical application of 1 mu l of juvenile hormone diluted in hexane, in the concentration of 2 mu g/mu l. Two controls were used; one control received no treatment (group C1) and other received topical application of 1 mu l of hexane (group C2). The aspect of the glandular cells, in not treated newly emerged workers, showed that they are not yet secreting actively. Cellular modifications happened according to the worker age and to the glandular area considered. The most active phase of the gland happened from the emergence to the 14th day. At the 25th day the cells had already lost their secretory characteristic, being the distal area the first to suffer degeneration. The treatment with juvenile hormone and hexane altered the temporal sequence of the glandular cycle, forwarding the secretory cycle and degeneration of the venom gland.

Morphological modifications induced by an artificial diet on the hypopharyngeal glands of Apis mellifera (Hymenoptera, apidae) during their degenerative process

The morphological aspects of the hypopharyngeal glands were analyzed in worker bees of Apis mellifera of 15 and 30 days of age. The individuals were kept in a room with controlled temperature at 32degreesC where they received water and either a protein or a high energy food. Nurse and foraging workers were used as a control for the experiment. The morphological results showed that the different diets modified the cell death characteristics and intensified its occurrence. Both diets caused precocious glandular degeneration. However, this anticipation of cell death was more pronounced in the glandular tissue of the workers who received the high energy diet when compared to the glands of the bees fed with the protein meal.The degenerative signs observed were an intense cytoplasmic vacuolization, with a loss of cytoplasm and of the cell boundaries, dilation or condensation of the cells and nuclei, and nuclear fragmentation. At the end of the degenerative process, we observed the extrusion of nuclei and, finally, the dissolution of the glands. The hypopharyngeal glands' remains were found in the haemolymph.

Effect of narcosis with CO2 on the ovarian development in queens of Apis mellifera (Hymenoptera, Apini)

Exposure of 1, 4, 7 & 10 day-old virgin queens of Apis mellifera Linne 1758 for 1 min to CO2 accelerated their ovarian development, having a similar effect as mating on the initial formation of the ovarian follicles. In 3 day-old queens the exposure to CO2 enhanced the initial stage of germ cell differentiation into oocytes and nurse cells in the ovarioles, a developmental stage only seen in 5 day-old untreated virgin queens, the age at which they are ready to mate. In 10 day-old untreated virgin queens, some regions of the ovarioles presented tissue disorganization and many cells with pycnotic nuclei. However, narcotized virgin queens of the same age did not present such ovary degeneration. Conversely, they showed nitid follicle formation, arising in the ovarioles' initial differentiation between nurse and oocytic chambers, although still without vitellogenesis. The accelerative effect of CO2 is limited to the ages near to those proper for mating, since 15 and 18 day-old treated virgin queens presented ovaries with extensive regions of high tissue disorganization and a great number of cells with pycnotic nuclei. According to the results, the narcosis presented three levels of effect on the ovary of honeybee virgin queens: 1) accelerated the germ cell differentiation, 2) preserved the tissue integrity even after the queen mating period and 3) stimulated the initial differentiation of a vitellarium. This later condition was only seen in untreated queens after mating. All these effects are not maintained if the queen is kept virgin beyond 15 days old.

Alterations induced by the juvenile hormone in glandular cells of the Apis mellifera venom gland: Applications on newly emerged workers (Hymenoptera, Apidae)

Histological and histochemical analyses were carried out in order to evaluate the influence of the topical application of a synthetic juvenile hormone on the secretory cycle and degeneration of the venom gland of Apis mellifera. Newly emerged workers received the topical application of synthetic hormone and the results were compared to the normal development of the secretory cycle in virgin and mated queens. The first worker group received the juvenile hormone diluted in hexane (2 mu g/mu L), the second received only mu L of hexane, and the third did not receive any kind of application. After the application the workers were returned to the colony and collected at the ages of 14 and 25 days of adult life. The groups with virgin queens and the other with mated queens, did not receive the treatment. The results show that the individuals treated with juvenile hormone and with pure hexane presented differences in the histological and cytochemical aspects of the secretory cells of the venom gland. The data indicate that both the juvenile hormone and hexane accelerate the activity of the secretory cycle and the degeneration of the venom gland; however, the juvenile hormone proved to be more effective than hexane. (c) 2006 Elsevier Ltd. All rights reserved.

Programmed cell death in the larval salivary glands of Apis mellifera (Hymenoptera, Apidae)

The morphological and histochemical features of degeneration in honeybee (Apis mellifera) salivary glands were investigated in 5th instar larvae and in the pre-pupal period. The distribution and activity patterns of acid phosphatase enzyme were also analysed. As a routine, the larval salivary glands were fixed and processed for light microscopy and transmission electron microscopy. Tissue sections were subsequently stained with haematoxylin-eosin, bromophenol blue, silver, or a variant of the critical electrolyte concentration (CEC) method. Ultrathin sections were contrasted with uranyl acetate and lead citrate. Glands were processed for the histochemical and cytochemical localization of acid phosphatase, as well as biochemical assay to detect its activity pattern. Acid phosphatase activity was histochemically detected in all the salivary glands analysed. The cytochemical results showed acid phosphatase in vesicles, Golgi apparatus and lysosomes during the secretory phase and, additionally, in autophagic structures and luminal secretion during the degenerative phase. These findings were in agreement with the biochemical assay. At the end of the 5th instar, the glandular cells had a vacuolated cytoplasm and pyknotic nuclei, and epithelial cells were shed into the glandular lumen. The transition phase from the 5th instar to the pre-pupal period was characterized by intense vacuolation of the basal cytoplasm and release of parts of the cytoplasm into the lumen by apical blebbing; these blebs contained cytoplasmic RNA, rough endoplasmic reticule and, occasionally, nuclear material. In the pre-pupal phase, the glandular epithelium showed progressive degeneration so that at the end of this phase only nuclei and remnants of the cytoplasm were observed. The nuclei were pyknotic, with peripheral chromatin and blebs. The gland remained in the haemolymph and was recycled during metamorphosis. The programmed cell death in this gland represented a morphological form intermediate between apoptosis and autophagy.

Action of permethrin on Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) semi-engorged females: Morpho-physiological evaluation of salivary glands

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cytotoxic effects of permethrin on mouse liver and spleen cells

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cytotoxic effects of permethrin in salivary glands of Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) semi-engorged females

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)