Cardiac mast cell proteases do not contribute to the regulation of the rat coronary vascular responsiveness to arterial delivered angiotensin I and II

Cardiac mast cells (MC) are apposed to capillaries within the heart and release renin and proteases capable of metabolizing angiotensins (Ang). Therefore, we hypothesized that mast cell degranulation could alter the rat coronary vascular responsiveness to the arterial delivered Ang I and Ang II, taking into account carboxypeptidase and chymase-1 activities. Hearts from animals that were either pretreated or not with systemic injection of the secretagogue compound 48/80 were isolated and mounted on a Langendorff apparatus to investigate coronary reactivity. The proteolytic activity of the cardiac perfusate from isolated hearts, pretreated or not with the secretagogue, toward Ang I and tetradecapeptide renin substrate was analyzed by HPLC. Coronary vascular reactivity to peptides was not affected by compound 48/80 pretreatment, despite the extensive amount of cardiac MC degranulation. Cardiac MC activation did not modify the generation of both Ang II and Ang 5-10 from Ang I by cardiac perfusate, activities that could be ascribed to MC carboxypeptidase and chymase-1, respectively. An aliskiren-resistant Ang I-forming activity was increased in perfusates from secretagogue-treated hearts. Thus, cardiac MC proteases capable of metabolizing angiotensins do not affect rat coronary reactivity to arterial delivered Ang I and II. (C) 2010 Elsevier Inc. All rights reserved.

Low-intensity Treadmill Exercise-related Changes in the Rat Stellate Ganglion Neurons

Stellate ganglion (SG) represents the main sympathetic input to the heart. This study aimed at investigating physical exercise-related changes in the quantitative aspects of SG neurons in treadmill-exercised Wistar rats. By applying state-of-the-art design-based stereology, the SG volume, total number of SG neurons, mean perikaryal volume of SG neurons, and the total volume of neurons in the whole SG have been examined. Arterial pressure and heart rate were also measured at the end of the exercise period. The present study showed that a low-intensity exercise training program caused a 12% decrease in the heart rate of trained rats. In contrast, there were no effects on systolic pressure, diastolic pressure, or mean arterial pressure. As to quantitative changes related to physical exercise, the main findings were a 21% increase in the fractional volume occupied by neurons in the SG, and an 83% increase in the mean perikaryal volume of SG neurons in treadmill-trained rats, which shows a remarkable neuron hypertrophy. It seems reasonable to infer that neuron hypertrophy may have been the result of a functional overload imposed on the SG neurons by initial posttraining sympathetic activation. From the novel stereological data we provide, further investigations are needed to shed light on the mechanistic aspect of neuron hypertrophy: what role does neuron hypertrophy play? Could neuron hypertrophy be assigned to the functional overload induced by physical exercise? (C) 2008 Wiley-Liss, Inc.

Effects of type II pyrethroid cyhalothrin on rat innate immunity: A flow cytometric study

Synthetic type II pyrethroids induce anxiety, immunosuppresion or, alternatively, immunostimulatory effects in laboratory animals. Macrophages and neutrophils are known to be key elements in cellular immune responses. The present study was designed to investigate the in vivo effects of cyhalothrin (1.0 and 3.0 mg/kg/once daily for 7 days) on macrophage and neutrophil activities, using a flow cytometry method. Results showed that cyhalothrin treatment decreased the percentage and intensity of phagocytosis performed by macrophages, but did not alter these parameters in neutrophils: and also decreased basal neutrophil oxidative burst and increased S. aureus-induced neutrophil oxidative burst, but did not alter these responses in macrophages. The present results are discussed in the light of a possible indirect action of cyhalothrin on macrophage and neutrophil activities via hypothalamic pituitary adrenal (HPA) axis activation. A possible direct effect of cyhalothrin on macrophage and neutrophil activities is also considered. (C) 2008 Elsevier B.V. All rights reserved.

Insulin Affects Tissue Organization and the Kinetics of Epithelial Cell Death in the Rat Ventral Prostate After Castration

Prostate growth and physiology are regulated by steroid hormones and modulated by multiple endocrine factors We investigated the action of insulin on the tissue organization and kinetics of epithelial cells in the rat ventral prostate (VP) in response to castration up to 120 hours after surgery by using an acute protocol of alloxan induced diabetes Diabetes caused a reduction in volume density (Vv(o)/) and volume of the epithelium The effects of castration on the epithelium were accelerated in the diabetic animals as determined by changes in V(o)/, and volume The smooth muscle cells became atrophic and apparently relaxed in response to castration in contrast to the spinous aspect observed in nondiabetic castrated rats Counting of apoptotic nuclei in the epithelium showed the classical apoptosis peak at 72 hours in nondiabetic rats and an advance of the apoptosis peak to 48 hours after castration in diabetic rats Insulin restored the time of the peak to 72 hours These results were confirmed after immunostaining for cleaved caspase 3 and suggest a survival and antiapoptotic effect on VP epithelial cells in both the presence and absence of androgen stimulation This idea is supported by the observation that insulin also reduced the overall rate of apoptosis at all experimental points analyzed before and after castration

Ultrastructure of Motor Nerve Terminals in the Anterior Third of Wistar Rat Tongue

The nerve terminals of intrinsic muscular fibers of the tongue of adult wistar rats was studied by using silver impregnation techniques, transmission electron microscopy (TEM), and high resolution scanning electron microscopy (HRSEM) to observe the nerve fibers and their terminals. Silver impregnation was done according to Winkelman and Schmit, 1957. For TEM, small blocks were fixed in modified Karnovsky solution, postfixed in 1% buffered osmium tetroxide solution, and embedded in Spurr resin. For HRSEM, the parts were fixed in 2% osmium tetroxide solution with 1/15 M sodium phosphate buffer (pH 7.4) at 4 degrees C for 2 h, according to the technique described by Tanaka, 1989. Thick myelinated nerve bundles were histologically observed among the muscular fibers. The intrafusal nerve fiber presented a tortuous pathway with punctiform terminal axons in clusters contacting the surface of sarcolemma. Several myelinated nerve fibers involved by collagen fibers of the endoneurium were observed in HRSEM in three-dimensional aspects. The concentric lamellae of the myelin sheath and the axoplasm containing neurofilaments interspersed among the mitochondria were also noted. In TEM, myofibrils, mitochondria, rough endoplasmic reticulum, Golgi`s apparatus, and glycogen granules were observed in sarcoplasm. It is also noted that the sarcomeres constituted by myofilaments with their A, I, and H bands and the electron dense Z lines. In areas adjacent to muscular fibers, there were myelinated and unmyelinated nerve fibers involved by endoneurium and perineurium. In the region of the neuromuscular junction, the contact with the sarcolemma of the muscular cell occurs forming several terminal buttons and showing numerous evaginations of the cell membrane. In the terminal button, mitochondria and numerous synaptic vesicles were observed. Microsc. Res. Tech. 72:464-470, 2009. (C) 2009 Wiley-Liss. Inc.

Laser phototherapy effect on protein metabolism parameters of rat salivary glands

The aim of this study was to evaluate the effects of infrared diode laser phototherapy (LP) on tissues of the submandibular gland (SMG) and parotid gland (PG). Wistar rats were randomly divided into experimental (A and B) and control (C) groups. A diode laser, 808 nm wavelength, in continuous wave mode, was applied to the PG, SMG and sublingual gland in the experimental groups on two consecutive days. The doses were 4 J/cm(2) and 8 J/cm(2), and total energy was 7 J and 14 J, respectively. The power output (500 mW) and power density (277 mW/cm(2)) were the same for both experimental groups. In order to visualize the area irradiated by the infrared laser, we used a red pilot beam (650 nm) with 3 mW maximum power for the experimental groups. For the control group, the red pilot beam was the only device used. The SMG and PG were removed after 1 week of the first irradiation. Total protein concentration, amylase, peroxidase, catalase and lactate dehydrogenase assays were performed, as well as histological analysis. Statistical tests revealed significant increase in the total protein concentration for groups A and B in the parotid glands (P < 0.05). Based on the results of this study, LP altered the total protein concentration in rats` parotid glands.

Immunocytochemical detection of dentine matrix protein 1 in experimentally induced reactionary and reparative dentine in rat incisors

Objective: Although the general mechanisms of dentinogenesis are understood, several aspects regarding tertiary dentine formation still deserve investigation, especially regarding the presence and distribution of some noncollagenous matrix proteins. As dentine matrix protein 1 (DMP 1) is present in primary dentine, it is possible that this protein may also be present in the dentine matrix secreted after injury, but there are no immunocytochemical studies attempting its detection in tertiary dentine. The aim of this study was to examine the ultrastructural immunolocalization of DMP 1 in the tertiary dentine after extrusion of the rat incisor. Study design: Upper incisors were extruded 3 mm and then repositioned into their sockets. After several periods, the incisors were fixed and processed for transmission electron microscopy and for immunocytochemistry for DMP 1. Results: Extrusion yielded both types of tertiary dentine, which varied in aspect and related cells. DMP 1 was found in the mineralized matrix of all types of dentine, presenting high affinity for collagen, but rare colloidal gold particles over predentine. DMP 1 was evident in the supranuclear region and inside the nucleus of some odontoblast-like cells. Conclusion: The observed association between DMP 1 and collagen seem to be essential for reactionary and reparative dentine formation. (C) 2010 Elsevier Ltd. All rights reserved.

Expression of placental glutathione S-transferase in rat tongue mucosa exposed to cigarette smoke

Glutatione S-transferases (GSTs) are a family of enzymes involved in detoxification of xenobiotics. Placental GST, known as GST-P, has been detected in tissues following exposure to carcinogenic agents being regarded a reliable biomarker of exposure and susceptibility in early phases of carcinogenesis. The aim of this study was to investigate the expressivity of GST-P positive foci in the rat tongue mucosa exposed to cigarette smoke by means of immunohistochemistry. A total of twelve male Wistar rats were distributed into two groups: negative control and experimental group exposed to cigarette smoke during 75 days. After experimental period, no histopathological changes in the tongue mucosa were evidenced in the negative control and the experimental group. However, a total of five GST-P positive foci were detected in two out of six animals exposed to cigarrette smoke. None control animals were noticed GST-P positive foci. These data indicate that expression of GST-P may reflect the carcinogenic effect of cigarette smoke as well as the genetic susceptibility of animals in relation to continuous carcinogens exposure.

Biocompatibility evaluation of alendronate paste in rat`s subcutaneous tissue

Alendronate is a known inhibitor of root resorption and the development of alendronate paste would enhance its utilization as intracanal medication. Therefore, this study aimed to investigate the biocompatibility of experimental alendronate paste in subcutaneous tissue of rats, for utilization in teeth susceptible to root resorption. The study was conducted on 15 male rats, weighing similar to 180-200 grams. The rats` dorsal regions were submitted to one incision on the median region and, laterally to the incision, the subcutaneous tissue was raised and gently dissected for introduction of two tubes, in each rat. The tubes were sealed at one end with gutta-percha and taken as control. The tubes were filled with experimental alendronate paste. The animals were killed at 7, 15 and 45 days after surgery and the specimens were processed in laboratory. The histological sections were stained with hematoxylin-eosin and analyzed by light microscopy. Scores were assigned to the in. ammatory process and statistically compared by the Tukey test (P < 0.05). Alendronate paste promoted severe inflammation process at 7 days, with statistically significant difference compared to the control (P < 0.05%). However, at 15 days, there was a regression of in. ammation and the presence of connective tissue with collagen fibers, fibroblasts and blood vessels was observed. After 45 days, it was observed the presence of well-organized connective tissue, with collagen fibers and fibroblasts, and few in. ammatory cells. No statistical difference was observed between the control and experimental paste at 15 and 45 days. The experimental alendronate paste was considered biocompatible with subcutaneous tissue of rat.

Immunohistochemical approach reveals involvement of inducible nitric oxide synthase in rat late development

To better understand the role of nitric oxide (NO) in mammal development, specifically in the transition of the fetal stages at birth, we studied the timing of cell-specific expression of inducible NO synthase (iNOS) isoform during gestational periods of rats, mainly at the late stages of intra-uterine development. Before experimentation, the samples were collected (from 17th to 21st gestational days), fixed in 10% buffered formalin and embedded in paraffin for histological procedures. Hereafter, the sections (5 mu m thickness) obtained from different embryos were immunostained by avidin-biotin-immunoperoxidase technique, by using antibody against iNOS isoform. The most of cell immunopositive was suggestive of granulocyte-like cells and those cells were resident close to the blood vessels in different organs, such as: lung, liver or bone marrow environment. Sometimes we noted immunopositive cells in the blood flow, as reported in the thymus. In agreement, iNOS expression, obtained by western blotting analysis, showed the same profile. Together, our data shows that iNOS expression increased gradually during the late stages of rat development (from E17 to E21) and it was executed by cells close to blood vessels. Thus, we can clearly to predict that this expression was finely modulated and it contributes for time-line dependent NO production during rat late development.

Expression of matrix metalloproteinases-2 and-9 and RECK during alveolar bone regeneration in rat

MMPs are endopeptidases that play a pivotal role in ECM turnover. RECK is a single membrane-anchored MMP-regulator. Here, we evaluated the temporal and spatial expression of MMP-2, MMP-9, and RECK during alveolar bone regeneration. The maxillary central incisor of Wistar rats was extracted and the animals were killed at 1, 3, 7, 10, 14, 21, 28, and 42 days post-operatively (n = 3/period). The hemimaxillae were collected, demineralized and embedded in paraffin. Immunohistochemical analysis was performed by the immunoperoxidase technique with polyclonal antibodies. On day 1, polymorphonuclear cells in the blood clot presented mild immunolabeling for MMPs. During bone remodeling, osteoblasts facing new bone showed positive staining for gelatinases and RECK in all experimental periods. MMPs were also found in the connective tissue and endothelial cells. Our results show for the first time that inactive and/or active forms of MMP-2, MMP-9 and RECK are differentially expressed by osteogenic and connective cells during several events of alveolar bone regeneration. This may be important for the replacement of the blood clot by connective tissue, and in the formation, maturation and remodeling of new bone.

Growth hormone induces bone morphogenetic proteins and bone-related proteins in the developing rat periodontium

The hypothesis that growth hormone (GH) up-regulates the expression of enzymes, matrix proteins, and differentiation markers involved in mineralization of tooth and bone matrices was tested by the treatment of Lewis dwarf rats with GH over 5 days, The molar teeth and associated alveolar bone were processed for immunohistochemical demonstration of bone morphogenetic proteins 2 and 4 (BMP-2 and -4), bone morphogenetic protein type IA receptor (BMPR-IA), bone alkaline phosphatase (ALP), osteocalcin (OC), osteopontin (OPN), bone sialoprotein (BSP), and E11 protein (E11), The cementoblasts, osteoblasts, and periodontal ligament (PDL) cells responded to GH by expressing BMP-2 and -4, BMPR-IA, ALP, OC, and OPN and increasing the numbers of these cells. No changes were found in patterns of expression of the late differentiation markers BSP and E11 in response to GH, Thus, GH evokes expression of bone markers of early differentiation in cementoblasts, PDL cells, and osteoblasts of the periodontium. We propose that the induction of BMP-2 and -4 and their receptor by GH compliments the role of GH-induced insulin-like growth factor 1 (IGF-1) in promoting bone and tooth root formation.

Rat forming incisor requires a rigorous ECM remodeling modulated by MMP/RECK balance

Reversion-inducing-cysteine-rich protein with Kazal motifs (RECK) is a single membrane-anchored MMP-regulator and regulates matrix metalloproteinases (MMP) 2, 9 and 14. In turn, MMPs are endopeptidases that play a pivotal role in remodeling ECM. In this work, we decided to evaluate expression pattern of RECK in growing rat incisor during, specifically focusing out amelogenesis process. Based on different kinds of ameloblasts, our results showed that RECK expression was conducted by secretory and post-secretory ameloblasts. At the secretory phase, RECK was localized in the infra-nuclear region of the ameloblast, outer epithelium, near blood vessels, and in the stellate reticulum. From the transition to the maturation phases, RECK was strongly expressed by non-epithelial immuno-competent cells (macrophages and/or dendritic-like cells) in the papillary layer. From the transition to the maturation stage, RECK expression was increased. RECK mRNA was amplified by RT-PCR from whole enamel organ. Here, we verified the presence of RECK mRNA during all stages of amelogenesis. These events were governed by ameloblasts and by non-epithelial cells residents in the enamel organ. Concluding, we found differential expression of MMPs-2, -9 and RECK in the different phases of amelogenesis, suggesting that the tissue remodeling is rigorously controlled during dental mineralization.

High doses of dexamethasone induce increased beta-cell proliferation in pancreatic rat islets

Rafacho A, Cestari TM, Taboga SR, Boschero AC, Bosqueiro JR. High doses of dexamethasone induce increased beta-cell proliferation in pancreatic rat islets. Am J Physiol Endocrinol Metab 296: E681-E689, 2009. First published January 21, 2009; doi:10.1152/ajpendo.90931.2008.-Activation of insulin signaling and cell cycle intermediates is required for adult beta-cell proliferation. Here, we report a model to study beta-cell proliferation in living rats by administering three different doses of dexamethasone (0.1, 0.5, and 1.0 mg/kg ip, DEX 0.1, DEX 0.5, and DEX 1.0, respectively) for 5 days. Insulin sensitivity, insulin secretion, and histomorphometric data were investigated. Western blotting was used to analyze the levels of proteins related to the control of beta-cell growth. DEX 1.0 rats, which present moderate hyperglycemia and marked hyperinsulinemia, exhibited a 5.1-fold increase in beta-cell proliferation and an increase (17%) in beta-cell size, with significant increase in beta-cell mass, compared with control rats. The hyperinsulinemic but euglycemic DEX 0.5 rats also showed a significant 3.6-fold increase in beta-cell proliferation. However, DEX 0.1 rats, which exhibited the lowest degree of insulin resistance, compensate for insulin demand by improving only islet function. Activation of the insulin receptor substrate 2/phosphatidylinositol 3-kinase/serine-threoninekinase/ribosomalprotein S6 kinase pathway, as well as protein retinoblastoma in islets from DEX 1.0 and DEX 0.5, but not in DEX 0.1, rats was also observed. Therefore, increasing doses of dexamethasone induce three different degrees of insulin requirement in living rats, serving as a model to investigate compensatory beta-cell alterations. Augmented beta-cell mass involves beta-cell hyperplasia and, to a lower extent, beta-cell hypertrophy. We suggest that alterations in circulating insulin and, to a lesser extent, glucose levels could be the major stimuli for beta-cell proliferation in the dexamethasone-induced insulin resistance.

Characterization of a Local Renin-Angiotensin System in Rat Gingival Tissue

Background: The systemic renin-angiotensin system (RAS) promotes the plasmatic production of angiotensin (Ang) II, which acts through interaction with specific receptors. There is growing evidence that local systems in various tissues and organs are capable of generating angiotensins independently of circulating RAS. The aims of this study were to investigate the expression and localization of RAS components in rat gingival tissue and evaluate the in vitro production of Ang II and other peptides catalyzed by rat gingival tissue homogenates incubated with different Ang II precursors. Methods: Reverse transcription - polymerase chain reaction assessed mRNA expression. Immunohistochemical analysis aimed to detect and localize renin. A standardized fluorimetric method with tripeptide hippuryl-histidyl-leucine was used to measure tissue angiotensin-converting enzyme (ACE) activity, whereas high performance liquid chromatography showed products formed after the incubation of tissue homogenates with Ang I or tetradecapeptide renin substrate (TDP). Results: mRNA for renin, angiotensinogen, ACE, and Ang II receptors (AT(1a), AT(1b), and AT(2)) was detected in gingival tissue; cultured gingival fibroblasts expressed renin, angiotensinogen, and AT(1a) receptor. Renin was present in the vascular endothelium and was intensely expressed in the epithelial basal layer of periodontally affected gingival tissue. ACE activity was detected (4.95 +/- 0.89 nmol histidyl-leucine/g/minute). When Ang I was used as substrate, Ang 1-9 (0.576 +/- 0.128 nmol/mg/minute), Ang II (0.066 +/- 0.008 nmol/mg/minute), and Ang 1-7 (0.111 +/- 0.017 nmol/mg/minute) were formed, whereas these same peptides (0.139 +/- 0.031, 0.206 +/- 0.046, and 0.039 +/- 0.007 nmol/mg/minute, respectively) and Ang 1 (0.973 +/- 0.139 nmol/mg/minute) were formed when TDP was the substrate. Conclusion: Local RAS exists in rat gingival tissue and is capable of generating Ang II and other vasoactive peptides in vitro. J Periodontol 2009;80:130-139.