Cloning, sequencing and expression analysis of the equine hepcidin gene by real-time PCR

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

Characteristics of seminal plasma proteins and their correlation with canine semen analysis

The objectives were to separate canine seminal plasma proteins (with SDS-PAGE) and to determine the correlation between specific proteins and semen characteristics. Three ejaculates from 20 mixed-breed dogs, of unknown fertility, were collected by digital manipulation. Ejaculate volume and color, sperm motility, sperm vigor, percentage of morphologically normal spermatozoa, and membrane integrity (hypoosmotic swelling test and fluorescent staining) were assessed. For each dog, seminal plasma was pooled from all three ejaculates and proteins were separated with SDS-PAGE, using polyacrylamide concentrations of 13% and 22% in the separation gels. After staining, gel images were digitized to estimate molecular weights (MW) and integrated optical density (IOD) of each lane and of individual bands. Total seminal plasma protein concentration was 2.19 +/- 1.56 g/dL (mean +/- SD; range 1.12-5.19 g/dL). A total of 37 protein bands were identified (although no dog had all 37 bands). In the 13% gel, molecular weights ranged from 100.6 to 17.1 kDa, with four bands (49.7, 33.2, 26.4, and 19.5 kDa) present in samples from all dogs. In the 22% gel, molecular weights ranged from 15.6 to 3.6 kDa, with nine bands (15.6, 13.5, 12.7, 11.7, 10.5, 8.7, 7.8, 5.6, and 4.9 kDa) present in samples from all dogs. Combined for both gels, the majority of bands (85%) had molecular weights < 17 kDa, with B20 (15.6 kDa) in high concentrations in samples from all dogs. There were positive correlations (P <= 0.01) between two bands, 134 (67 kDa) and B5 (58.6 kDa), and sperm motility (r = 0.66 and r = 0.46), sperm vigor (r = 0.56 and r = 0.66), percentage of morphologically normal spermatozoa (r = 0.55 and r = 0.59), the hypoosmotic swelling test (r = 0.76 and r 0.68), and fluorescent staining (r = 0.56 and r = 0.59), respectively. In conclusion, 37 proteins were identified in seminal plasma; two were significantly correlated with semen characteristics. (c) 2007 Elsevier B.V. All rights reserved.

PAR(2) and Temporomandibular Joint Inflammation in the Rat

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

Bone healing in critical-size defects treated with platelet-rich plasma: a histologic and histometric study in the calvaria of diabetic rat

Objective. The objective of this study was to histologically analyze the influence of the platelet-rich plasma (PRP) on bone healing in surgically created critical-size defects (CSD) in the calvaria of diabetic rats.Study design. A total of 20 diabetic rats were divided into 2 groups: C (control) and PRP. A 5-mm diameter CSD was created in the calvarium of each animal. In Group C, the defect was filled by blood clot only. In Group PRP, 0.35 mL of PRP was placed in the defects. All animals were humanely killed 30 days postoperatively. Histometric and histologic analyses were performed. Data were statistically analyzed (t test, P < .05).Results. No defect completely regenerated with bone. Group PRP had a statistically greater amount of bone formation than Group C (37.22% +/- 6.00% and 21.68% +/- 11.35%, respectively).Conclusion. PRP placed in the defects significantly enhanced bone healing in CSD in the calvaria of diabetic rats both qualitatively and quantitatively. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010; 109: 72-78)

Histomorphometric analysis of tissue responses to bioactive glass implants in critical defects in rat calvaria

The aim of this study was to evaluate the osteogenic behavior of two chemically similar bioactive glass products (Biogran (R) and Perioglas (R)) implanted in critical bone defects in rat calvaria. Thirty-six transfixed bone defects of 8 mm diameter were made surgically in adult male Wistar rats. The animals were distributed equally into three groups: Biogran (GI), Perioglas (GII) and without implant material (control; GIII). The morphology and composition of both bioactive glasses were analyzed by scanning electron microscopy and energy-dispersive spectrometry. Tissue specimens were analyzed at the biological time points of 15, 30 and 60 days by optical microscopy and morphometry, demonstrating biocompatibility for the tested materials with moderate chronic inflammation involving their particles. Bone neoformation resulted only as a reparative reaction to an intentionally produced defect and was limited to the defect's edges. No statistically significant differences among the groups were observed. At the scar interstice, abundant deposits of collagenous fibers enveloping the particles were noted. The present results indicated that the bioactive glasses, under the experimental conditions analyzed, did not show osteogenic behavior. Copyright (c) 2007 S. Karger AG, Basel.

Bioglass Associated With Leukocyte-Poor Platelet-Rich Plasma in the Rabbit Maxillary Sinus: Histomorphometric, Densitometric, and Fractal Analysis

The objective of the present study was to evaluate the outcomes of autogenous bone graft (AB) and bioglass (BG) associated or not with leukocyte-poor platelet-rich plasma (LP-PRP) in the rabbit maxillary sinus (MS) by histomorphometric and radiographic analysis. Twenty rabbits divided into 2 groups (G1, G2) were submitted to sinus lift surgery. In G1, 10 MS were grafted with AB and 10 MS were grafted with BG. In G2, 10 MS were grafted with AB + LP-PRP and 10 MS were grafted with BG + LP-PRP. After 90 days, the animals were killed and specimens were obtained, x-rayed, and submitted to histomorphometric, radiographic bone density (RD) and fractal dimension analysis. Radiographic bone density mean values (SD), expressed as aluminum equivalent in mm, of AB, BG, AB + LP-PRP, and BG + LP-PRP groups were 1.79 (0.31), 2.04 (0.39), 1.61 (0.28), and 1.53 (0.30), respectively. Significant differences (P < 0.05) were observed between BG and AB, and BG + PRP and BG. Fractal dimension mean values were 1.48 (0.04), 1.35 (0.08), 1.44 (0.04), and 1.44 (0.06), respectively. Significant differences were observed between BG and AB, and AB + LP-PRP and BG. Mean values for the percentage of bone inside MS were 63.30 (8.60), 52.65 (10.41), 55.25 (7.01), and 51.07 (10.25), respectively. No differences were found. No correlations were observed among percentage of bone, RD and FD. Histological analysis showed that MS treated with AB presented mature and new bone formation. The other groups showed minor bone formation. Within the limitations of this study, the results indicated that at a 90-day time end point, AB yielded better results than AB + LP-PRP, BG, and BG + LP-PRP and should be considered the primary material for MS augmentation.

Histomorphometric analysis of the repair process of autogenous bone grafts fixed at rat calvaria with cyanoacrylate

Objective: The purpose of this study was to perform histological and histometric analyses of the repair process of autogenous bone grafts fixed at rat calvaria with ethyl-cyanoacrylate adhesive. Material and Methods: Thirty-two rats were divided into two groups (n=16), Group I - Control and Group II - Adhesive. Osteotomies were made at the right parietal bone for graft obtainment using a 4-mm-diameter trephine drill. Then, the bone segments were fixed with the adhesive in the parietal region of the opposite side to the donor site. After 10 and 30 days, 8 animals of each group were euthanized and the calvarias were laboratorially processed for obtaining hematoxylin and eosin-stained slides for histological and histometric analyses. Results: An intense inflammatory reaction was observed at the 10-day period. At 30 days, this reaction was less intense, despite the presence of adhesive at the recipient-site/graft interface. Graft incorporation to the recipient site was observed only at the control group, which maintained the highest graft size at 10 and 30 days. Conclusions: Although the fragment was stable, the presence of adhesive in Group II did not allow graft incorporation to the recipient site, determining a localized, discrete and persistent inflammatory reaction.

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.

IGFR-I expression and structural analysis of the hard palatine mucosa in an ethanol-drinking rat strain (UChA and UChB)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Cardiac remodeling in a rat model of diet-induced obesity

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

Chronic caffeine intake increases androgenic stimuli, epithelial cell proliferation and hyperplasia in rat ventral prostate

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

Influence of acute pancreatitis on the in vitro responsiveness of rat mesenteric and pulmonary arteries

Background: Acute pancreatitis is an inflammatory disease characterized by local tissue injury and systemic inflammatory response leading to massive nitric oxide (NO) production and haemodynamic disturbances. Therefore, the aim of this work was to evaluate the vascular reactivity of pulmonary and mesenteric artery rings from rats submitted to experimental pancreatitis.Male Wistar rats were divided into three groups: saline (SAL); tauracholate (TAU) and phospholipase A(2) (PLA(2)). Pancreatitis was induced by administration of TAU or PLA(2) from Naja mocambique mocambique into the common bile duct of rats, and after 4 h of duct injection the animals were sacrificed. Concentration-response curves to acetylcholine (ACh), sodium nitroprusside (SNP) and phenylephrine (PHE) in isolated mesenteric and pulmonary arteries were obtained. Potency (pEC(50)) and maximal responses (E(MAX)) were determined. Blood samples were collected for biochemical analysis.Results: In mesenteric rings, the potency for ACh was significantly decreased from animals treated with TAU (about 4.2-fold) or PLA(2) (about 6.9-fold) compared to saline group without changes in the maximal responses. Neither pEC(50) nor E(MAX) values for Ach were altered in pulmonary rings in any group. Similarly, the pEC(50) and the E(MAX) values for SNP were not changed in both preparations in any group. The potency for PHE was significantly decreased in rat mesenteric and pulmonary rings from TAU group compared to SAL group (about 2.2- and 2.69-fold, for mesenteric and pulmonary rings, respectively). No changes were seen in the E(MAX) for PHE. The nitrite/nitrate (NO(x)(-)) levels were markedly increased in animals submitted to acute pancreatitis as compared to SAL group, approximately 76 and 68% in TAU and PLA(2) protocol, respectively.Conclusion: Acute pancreatitis provoked deleterious effects in endothelium-dependent relaxing response for ACh in mesenteric rings that were strongly associated with high plasma NO(x)(-) levels as consequence of intense inflammatory responses. Furthermore, the subsensitivity of contractile response to PHE in both mesenteric and pulmonary rings might be due to the complications of this pathological condition in the early stage of pancreatitis.

An immunocytochemical and in situ hybridization analysis of annexin 1 expression in rat mast cells: Modulation by inflammation and dexamethasone

The presence and localization of the anti-inflammatory protein annexin 1 (also known as lipocortin 1) in perivenular rat mast cells was investigated here. Using the rat mesenteric microvascular bed and a combination of morphologic techniques ranging from immunofluorescence to electron microscopy analyses, we detected the presence of annexin 1 in discrete intracellular sites, both in the nucleus and in the cytoplasm. In resting mast cells, most of the protein pool (approximately 80% of the cytosolic portion) was localized to cytoplasmic granules. In agreement with other cell types, treatment of rats with dexamethasone (0.2 mg/kg, ip) increased annexin 1 expression in mast cells, inducing a remarkable appearance of dusters of protein immunoreactivity. This effect was most likely the result of de novo protein synthesis as determined by an increase in mRNA seen by in situ hybridization. Triggering an ongoing experimental inflammatory response (0.3 mg of carrageenin, ip) increased annexin 1 mRNA and protein levels. In conclusion, we report for the first time the localization of annexin 1 in connective tissue mast cells, and its susceptibility not only to glucocorticoid hormone treatment, but also to an experimental acute inflammatory response.

Short-term stromal alterations in the rat ventral prostate following alloxan-induced diabetes and the influence of insulin replacement

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

High fat-induced obesity associated with insulin-resistance increases FGF-2 content and causes stromal hyperplasia in rat ventral prostate

Obesity affects sex hormone secretion, which can negatively influence prostatic structure, homeostasis, and disease. This investigation aimed to evaluate the repercussions of obesity induced by a high-fat diet on the rat prostate, with or without treatment with the aromatase inhibitor, Letrozole. Adult Wistar rats were fed a high-fat diet (20% saturated fat, O) for 15 weeks to induce obesity or received a balanced diet (4% fat, C). Then, a group of C and O rats were daily treated with Letrozole (1 mg/kg b.w. per day) for 2 weeks (CL and OL, respectively). Subsequently, ventral prostate was processed for analysis by transmission electron microscopy, immunohistochemistry, and Western blotting. Obesity decreased 70% of the testosterone plasma level. The prostate showed epithelial atrophy and dilated acini in the intermediate portion and epithelial wrinkling in the distal tips. The relative frequency of smooth muscle alpha-actin in the O group increased by 67%. Ultrastructurally, epithelial cells in obese animals presented altered secretory organelles, lipid droplets, and thicker subjacent fibromuscular layer. Letrozole treatment caused a partial restoration of the prostatic changes caused by obesity. Obesity increased the prostatic content of fibroblast growth factor-2 (FGF-2) by 150%, and Letrozole treatment increased this protein even more in the control and obese groups. This investigation shows that obesity provokes structural and ultrastructural changes in the epithelium of rat prostate; these changes might affect gland homeostasis and physiology. The epithelial and smooth muscle cell hyperplasia and increased FGF-2 expression observed in this experimental model of obesity/insulin-resistance might explain the high frequency of benign prostatic hyperplasia in insulin-resistant men.