Development of a rabbit's urethral sphincter deficiency animal model for anatomical-functional evaluation

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

Staining methods applied to glycol methacrylate embedded tissue sections

The use of glycol methacrylate (GMA) avoids some technical artifacts, which are usually observed in paraffin-embedded sections, providing good morphological resolution. on the other hand, weak staining have been mentioned during the use of different methods in plastic sections. In the present study, changes in the histological staining procedures have been assayed during the use of staining and histochemical methods in different GMA-embedded tissues.Samples of tongue, submandibular and sublingual glands, cartilage, portions of respiratory tract and nervous ganglion were fixed in 4% formaldehyde and embedded in glycol methacrylate. The sections of tongue and nervous ganglion were stained by H&E. Picrosirius, Toluidine Blue and Sudan Black B methods were applied, respectively, for identification of collagen fibers in submandibular gland, sulfated glycosaminoglycans in cartilage (metachromasia) and myelin lipids in nervous ganglion. Periodic Acid-Schiff (PAS) method was used for detection of glycoconjugates in submandibular gland and cartilage while AB/PAS combined methods were applied for detection of mucins in the respiratory tract. In addition, a combination of Alcian Blue (AB) and Picrosirius methods was also assayed in the sublingual gland sections.The GMA-embedded tissue sections showed an optimal morphological integrity and were favorable to the staining methods employed in the present study. In the sections of tongue and nervous ganglion, a good contrast of basophilic and acidophilic structures was obtained by H&E. An intense eosinophilia was observed either in the striated muscle fibers or in the myelin sheaths in which the lipids were preserved and revealed by Sudan Black B. In the cartilage matrix, a strong metachromasia was revealed by Toluidine Blue in the negatively-charged glycosaminoglycans. In the chondrocytes, glycogen granules were intensely positive to PAS method. Extracellular glycoproteins were also PAS positive in the basal membrane and in the region occupied by the lamina externa and reticular fibers surrounding each smooth muscle cells of the blood vessels. In the epithelial cells of the respiratory tract, acid and neutral mucins were histochemically detected by AB and PAS methods, respectively. Moreover, granules containing acid and neutral mucins were revealed in purple by AB and PAS concomitantly. In the sublingual gland sections, a distinct affinity of acid mucins by AB (in turquoise-blue) and collagen fibers by Picrosirius (in red) was obtained when these methods were combined. Although some routine dyes used in paraffin sections have showed a weak stain in historesin sections, our results showed that different dyes could be applied in GMA sections if modified staining procedures were assayed. Therefore, appropriate staining contrast and, thus, detection of one or different substances in a same section can be acquired in association to the good morphological resolution provided by GMA. (C) 2003 Elsevier Ltd. All rights reserved.

Alterações vasculares em ratos obesos por dieta rica em gordura: papel da Via L-arginina/NO Endotelial

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

Remodeling of rat ventral prostate after castration involves heparanase-1

Androgen deprivation causes the rat ventral prostate to reduce to 10% of its original size by 21 days after castration. The regressive changes result from the loss of epithelial cells by apoptosis and marked reorganization of the stroma. We have investigated whether these changes are accompanied by variations in heparanase expression. The ventral prostate of castrated rats was collected and processed for the quantification of heparan sulfate (HS), for the measurement of heparanase expression and its localization by reverse transcription/polymerase chain reaction, Western blotting, and immunohistochemistry, and for transmission electron microscopy (TEM). Absolute HS content decreased significantly as early as day 7 after surgery. Heparanase mRNA peaked 7 days after castration. The heparanase proenzyme (65 kDa) and the active form (50 kDa) were identified and peaked on day 7 after castration; this coincided with maximum HS-degrading activity. Heparanase was located to the basolateral surface of epithelial cells and in the adjacent stroma. After castration, staining for heparanase was reduced in the epithelium and increased in the stroma. TEM revealed that the peak of heparanase expression at day 7 after castration was associated with extensive changes in the basement membrane of the epithelium, endothelium and smooth muscle cells involving cell shrinkage and/or deletion by apoptosis. These results suggest that heparanase expression increases after castration and correlates with a decreased amount of HS. This variation in heparanase expression is involved in tissue remodeling and in the control of the regressive pattern after 1 week of androgen deprivation.

Expression and processing of recombinant sarafotoxins precursor in Pichia pastoris

Sarafotoxins are peptides isolated from the Atractaspisw snake venom. with strong constrictor effect on cardiac and smooth muscle. They are structurally and functionally related to endothelins. The sarafotoxins precursor cDNA predicts an unusual structure 'rosary-type', with 12 successive similar stretches of sarafotoxin (SRTX) and spacer, in the present work, the recombinant precursor of SRTXs was sub-cloned and expressed in the yeast Pichia pastoris. and secreted to the culture medium, Characterization by SDS-PAGE, immunoblot, mass spectrometry and biological activity, suggests that intact precursor was expressed but processing into mature toxins also occurred. Furthermore, our results indicate that the correct proportion of sarafotoxin types as contained in the precursor, is obtained in the yeast culture medium. Contractile effects of the expressed toxins, on rat and Bothrops jararaca isolated aorta, were equivalent to 5 X 10(-10) M and 5 x 10(-11) M of sarafotoxin b, respectively. The enzymes responsible for the complete maturation of sarafotoxins precursor are still unknown. Our results strongly suggest that the yeast Pichia pastoris is able to perform such a maturation process. Thus, the yeast Pichia pastoris may offer an alternative to snake venom gland to tentatively identify the molecular process responsible for SRTXs release. (C) 2001 Elsevier B.V. Ltd. All rights reserved.

Protonectin (1-6): A novel chemotactic peptide from the venom of the social wasp Agelaia pallipes pallipes

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

Effects of exercise training on the cardiovascular system: Pharmacological approaches

Physical exercise promotes beneficial health effects by preventing or reducing the deleterious effects of pathological conditions, such as arterial hypertension, coronary artery disease, atherosclerosis, diabetes mellitus, osteoporosis, Parkinson's disease, and Alzheimer disease. Human movement studies are becoming an emerging science in the epidemiological area and public health. A great number of studies have shown that exercise training, in general, reduces sympathetic activity and/or increases parasympathetic tonus either in human or laboratory animals. Alterations in autonomic nervous system have been correlated with reduction in heart rate (resting bradycardia) and blood pressure, either in normotensive or hypertensive subjects. However, the underlying mechanisms by which physical exercise produce bradycardia and reduces blood pressure has not been fully understood. Pharmacological studies have particularly contributed to the comprehension of the role of receptor and transduction signaling pathways on the heart and blood vessels in response to exercise training. This review summarizes and examines the data from studies using animal models and human to determine the effect of exercise training on the cardiovascular system. (c) 2007 Elsevier B.V. All rights reserved.

Enhanced airways responsiveness in rats depleted of sensory neuropeptides by neonatal capsaicin treatment

This study aimed to investigate the in vivo and in vitro reactivity of airway smooth muscle in rats depleted of sensory neuropeptides by treatment with capsaicin at neonatal stage. Wistar rats were neonatally injected with either capsaicin (50 mg/kg, s.c., 2nd day of life) or its vehicle (10% ethanol and 10% Tween 80, in 0.9% w/v NaCl solution) and used at adult ages (60-70 days later). Analysis of the lungs showed a higher number of infiltrating neutrophils, eosinophils and mononuclear cells into the peribronchiolar regions of capsaicin-pretreated rats compared to vehicle group. This was associated with a higher contraction index of bronchiolar wall in the capsaicin group. The in vitro tracheal reactivity in response to methacholine (full muscarinic agonist) and pilocarpine (partial muscarinic agonist) was also significantly higher in capsaicin-pretreated rats compared to vehicle group. In conclusion, the neuropeptide depletion by capsaicin neonatal treatment lead to marked contraction of the rat airways at adult age, suggesting a protective role for C fibers in the lungs. (C) 2003 Elsevier B.V. Ireland Ltd. All rights reserved.

Structure of the posthepatic septum and its influence on visceral topology in the tegu lizard, Tupinambis merianae (Teiidae : Reptilia)

The posthepatic septum (PHS) divides the body cavity of Tupinambis merianae into two parts: the cranial one containing the lungs and liver and the caudal one containing the remaining viscera. The PHS is composed of layers of collagenous fibers and bundles of smooth muscle, neither of which show systematic orientation, as well as isolated blood vessels, lymphatic vessels, and nerves. Striated muscle of the abdominal wall does not invade the PHS. The contractions of the smooth muscles may stabilize the pleurohepatic cavity under conditions of elevated aerobic needs rather than supporting breathing on a breath-by-breath basis. Surgical removal of the PHS changes the anatomical arrangement of the viscera significantly, with stomach and intestine invading the former pleurohepatic cavity and reducing the space for the lungs, Thus, the PHS is essential to maintain the visceral topography in Tupitionibis. J. Morphol. 258:151-157, 2003. (C) 2003 Wiley-Liss. Inc.

Tissue evidence of the testosterone role on the abnormal growth and aging effects reversion in the gerbil (Meriones unguiculatus) prostate

Prostate differentiation during embryogenesis and its further homeostatic state maintenance during adult life depend on androgens. Abundant biological data suggest that androgens play an important role in the development of the prostate cancer and other prostatic diseases. The objective of this work was to evaluate the effects of the testosterone supplementation in gerbil (a new experimental model) at different ages. Tissues from experimental animals were studied by histological and histochemistry procedures, androgen receptor immunohistochemistry assay, morphometric-stereological analysis, and transmission electron microscopy (TEM). After the treatment were observed increase of prostate weight and epithelium height in all ages studied. In some adult and aged treated animals, hyperplasic and displasic process were observed, including prostatic intraepithelial neoplasias and adenocarcinomas. Increase of the thickness of the smooth muscle cell (SMC) layer was observed in pubescent and adult animals and TEM revealed apparent SMC hypertrophy. An apparent increase in the frequency of blood vessels distributed by the subepithelial stroma in the treated animals was noticed. Reversion of the natural effects of aging on the prostate was observed in the aged treated animals in some acini of the gland. These data demonstrate that the gerbil prostate is susceptible to androgenic action at the studied ages and it can serve, for example, as experimental model to studies of prostate neoplasic process induction and hormonal therapy in aged animals.

Structure, histochemistry, and ultrastructure of the epithelium and stroma in the gerbil (Meriones unguiculatus) female prostate

The female prostate has aroused scientific interest because it is subjected to the same diseases compromising the male prostate during aging. The objective of this work was to characterize structurally, cytochemically, and ultrastructurally the tissue compartments of the normal adult female prostate of Meriones unguiculatus gerbils. The morphological analyses showed that the gerbil's female prostate is constituted of a cluster of glands and ducts inserted in a musculofibrous stroma. The alveolar epithelium is differentiated and consisted of basal proliferating cells, intermediary cells, and secretory cells. The secretory cells are the most numerous cell type and continuously secrete glycoproteins. The basal cells are the source of the secretory cells and they are then responsible for the alveolus renovation. The prostatic stroma is abundant and rich in elastic and collagen fibers, which are closely associated with smooth muscle cells and fibroblasts. The results showed that the gerbil's female prostate shows morphological and ultrastructural homology to the human female prostate (Skene's gland), and despite being a small organ, it is a mature and physiologically active gland. (C) 2003 Elsevier Ltd. All rights reserved.

Biological behavior of the gerbil ventral prostate in three phases of postnatal development

In this study, we characterized the gerbil's ventral prostate histology ultrastructurally and quantitatively throughout three phases of postnatal development (young, adult, and old) in order to comprehend its biological behavior and propensity to developing spontaneous lesions with aging. The gerbil prostate is composed of alveoli and ducts immersed in a stroma composed of smooth muscle, fibroblasts, collagen and elastic fibers and vessels. The prostate tissue components present morphological and quantitative aspects that vary according to age. Young animals have an immature gland with modest secretory activity. Synthetic activity remained stable in adult and old gerbil. However, prostatic morphology was altered in the aging, showing an increased epithelium and stromal fibrosis. The nuclei of the secretory cells increased with aging, whereas nucleoli presented few alterations during postnatal development. The epithelial proliferation and stromal remodeling noted in this study indicate that the gerbil prostate may respond to the androgen declines typical of senescence through epithelial proliferation and stromal remodeling.

Tissue alterations in the Guinea pig lateral prostate following antiandrogen flutamide therapy

The flutamide antiandrogenic effects oil the Guinea pig male prostate morphology in puberal, post-puberal and adult ages were evaluated in the present study. Daily-treated group animals received flutamide Subcutaneous injection at a dose of 10 mg/Kg body weight for 10 days. The control group animals received a pharmacological vehicle under the same conditions. The lateral prostate was removed, fixed and processed for light and transmission electron microscopy. The results revealed all increase of the acinus diameter in the treated puberal animals and straitness in the stromal compartment around the acini. The epithelial cells exhibited cubic phenotype. In the post-puberal and adult animals, a decrease of the acinus diameter was observed, as well as an increase of the smooth muscle layer and presence of the folds at epithelium. The ultrastructural evaluation of the secretory cells in the treated group demonstrated endomembrane enlargement, mainly in the rough endoplasmic reticulum and Golgi apparatus. In addition, a decrease of the microvilli and alterations in the distribution patterns and density of the stromal fibrillar components were observed. In Conclusion, the flutamide treatment exerts tissue effects oil the lateral prostate, promoting stroma/epithelium alterations.

Acid phosphatase activity in gerbil prostate: comparative study in male and female during postnatal development

The prostate is present in both male and female mammals. It is composed of secretory epithelium, connective stroma, smooth muscle and neuroendocrine cells, which are under hormonal regulation. Acid phosphatases catalyze the hydrolysis of orthophosphate monoesters. We have compared the expression of acid phosphatases in gerbil (Meriones unguiculatus) prostate glands in both sexes using young, adult and old animals. Eighteen prostates were isolated, frozen, sectioned, fixed, incubated with sodium beta-glycerophosphate sodium, washed with acetate buffer solution, treated with ammonium sulfide and counterstained with Methyl-Green aqueous solution. Ultracytochemical analyses were also conducted. This substrate revealed total acid phosphatase activity. The expression of the enzyme was heterogeneous, occurring in all ages during postnatal development. The data. revealed that the female prostate matured before the male prostate. In addition, acid phosphatase activity in both sexes was regulated by androgen variation concomitant with development. (C) 2004 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.

Postnatal growth of the ventral prostate in Wistar rats: A stereological and morphometrical study

Morphological and stereological analyses were used to characterize the growth kinetics of the Wistar rat ventral prostate (VP). Volume density and absolute volume of the epithelium, lumen, smooth muscle cells (SMCs), and nonmuscular stroma were determined by stereology and paired with plasma testosterone levels and different morphometric measurements. The VP shows an initial growth within the first 3 weeks, a resting phase, and the puberal growth. The puberal growth was coincident with the raise in plasma testosterone. Lumen formation occurred within the 3 postnatal weeks. After an expected increase during puberty, the lumen showed a further increase at the 12th week. The volume density of the nonmuscular stroma and of the SMCs decreased slowly postnatally. Absolute volume of the luminal compartment showed three phases of growth (weeks 1-3, 6-9, and 11-12). on the other hand, the increase in the absolute volume of the epithelium was steady up to the 8th week and then showed a marked increase up the 10th week. The increase in epithelial volume was characterized morphologically by the presence of epithelial infoldings and sprouts. The growth of the epithelium showed a 2-week delay as compared to the lumen and occurred only until the 10th week. The epithelial height was variable but could be related to the synthetic activity of the epithelium. In conclusion, the postnatal growth of the VP results from a combination of epithelial proliferation/differentiation and synthesis/accumulation of the secretory products in the lumen.