Pedro Duarte Novaes

Proatherosclerotic effects of chronic stress in male rats: Altered phenylephrine sensitivity and nitric oxide synthase activity of aorta and circulating lipids

The aim of this study was to analyze the effects of chronic mild unpredictable stress (CMS) on the vasoconstrictor response and morphology of the thoracic aorta and serum lipid profiles in rats. Male Sprague–Dawley rats were submitted to CMS, which consisted of the application of different stressors for 7 days per week across 3 weeks. The rats were sacrificed 15 days after CMS expsoure. CMS induced supersensitivity to the vasoconstrictor effect of phenylephrine in endothelium-intact thoracic aortic rings without changes in aortic rings without endothelium, or pre-incubated with nitric oxide (NO) synthesis inhibitor. Rats submitted to CMS showed hypertrophy of the intima and tunica media of thoracic aorta, increased serum levels of triglycerides, total cholesterol, very low-density lipoprotein cholesterol, low-density lipoprotein cholesterol and atherogenic index, without changes in high-density lipoprotein cholesterol levels, when compared with control rats. These data indicate that CMS induces physiological and morphological changes that may contribute to the development of atherosclerosis by mechanisms related to deficiency in NO production and dyslipidemia.

Nandrolone and resistance training induce heart remodeling Role of fetal genes and implications for cardiac pathophysiology

AIMS This study was conducted to assess the isolated and combined effects of nandrolone and resistance training on cardiac morphology, function, and mRNA expression of pathological cardiac hypertrophy markers. MAIN METHODS Wistar rats were randomly divided into four groups and submitted to 6 weeks of treatment with nandrolone and/or resistance training. Cardiac parameters were determined by echocardiography. Heart was analyzed for collagen infiltration. Real-time RT-PCR was used to assess the pathological cardiac hypertrophy markers. KEY FINDINGS Both resistance training and nandrolone induced cardiac hypertrophy. Nandrolone increased the cardiac collagen content, and reduced the cardiac index in non-trained and trained groups, when compared with the respective vehicle-treated groups. Nandrolone reduced the ratio of maximum early to late transmitral flow velocity in non-trained and trained groups, when compared with the respective vehicle-treated groups. Nandrolone reduced the alpha-myosin heavy chain gene expression in both non-trained and trained groups, when compared with the respective vehicle-treated groups. Training reduced the beta-myosin heavy chain gene expression in the groups treated with vehicle and nandrolone. Only the association between training and nandrolone increased the expression of the skeletal alpha-actin gene and atrial natriuretic peptide in the left ventricle. SIGNIFICANCE This study indicated that nandrolone, whether associated with resistance training or not, induces cardiac hypertrophy, which is associated with enhanced collagen content, re-expression of fetal genes the in left ventricle, and impaired diastolic and systolic function.

Immunolocalization of CSF-1, RANKL and OPG in the enamel-related periodontium of the rat incisor and their implications for alveolar bone remodeling

The enamel-related periodontium (ERP) in rat incisors is related to bone resorption. In these teeth the face of the socket related to the enamel is continuously removed at the inner side and newly formed at the outer side. CSF-1, RANKL and OPG are regulatory molecules essential for osteoclastogenesis. To verify the effects of impeded eruption on bone remodeling, the tooth eruption was prevented by immobilization of lower rat incisor and CSF-1, RANKL and OPG distribution in the ERP was analyzed after 18 days of immobilization and in normal eruption. The region of the alveolar crest of the rat incisor was used. Immunohistochemistry and tartrate-resistant acid phosphatase (TRAP) were performed. The immunostaining of the dental follicle was quantified using Leica QWin software. Positive-TRAP osteoclasts were counted, and both groups were compared. In the normal incisor, the number of osteoclasts was significantly greater than in the immobilized tooth. In the dental follicle, there was no significant difference in the immunostaining intensity for CSF-1 and OPG between the groups (p > 0.05), but for RANKL the immobilized incisor group showed immunostaining intensity smaller than the normal incisor group (p < 0.01). These findings suggest that changes in the ERP, in the immobilized incisor, modify the RANKL/OPG ratio, in the presence of CSF-1, altering the metabolism of cells that participate in the bone remodeling.

The effect of lead on the eruption rates of incisor teeth in rats

The effects of lead on the continuously erupting rat incisors under normo-, hyper- and hypofunctional conditions were investigated. Left lower incisors of 20 rats were rendered unimpeded (hypofunctional) by cutting them out of occlusion every 2 days; the right lower incisors of these rats were considered hyperfunctional. Measurements on normally growing teeth (normofunctional) were carried out in a group of ten rats whose teeth were not cut but only marked every 2 days. On day 7 of the experiment, half of the rats from these two groups were given a single intraperitoneal injection of lead acetate (40 mg/kg), and the other half received sodium acetate (22 mg/kg). Another group of 15 rats was used to obtain blood samples for lead determination 1 h, and 10, 20, and 30 days after lead administration. Animals were killed on day 32. Hypofunctional incisors from lead-treated rats erupted more slowly than control ones (P<0.05). These results show a previously unreported toxic effect of heavy metals.

Chronic stress, but not hypercaloric diet, impairs vascular function in rats

The aim of this study was to evaluate vascular and metabolic effects of chronic mild unpredictable stress (CMS) and hypercaloric diet (HD) without carbohydrate supplementation in rats. Male Sprague-Dawley rats were randomly assigned to four groups: Control, HD, CMS, and HD plus CMS. CMS consisted of the application of different stressors for 3 weeks. The rats were killed 15 days after CMS exposure. The HD group presented higher plasma lipid concentrations, without changes in fasting glucose concentration, glucose tolerance test, and vascular function and morphology, in comparison with the control group. Stressed rats presented higher fasting blood concentration of insulin, higher homeostasis model assessment index values and area under the curve in an oral glucose tolerance test, in comparison with non-stressed rats. CMS increased the plasma concentrations of corticosterone and lipids, and the atherogenic index values, without change in high-density lipoprotein level. CMS increased intima-media thickness and induced endothelium-dependent supersensitivity to phenylephrine, and lowered the relaxation response to acetylcholine in the thoracic aorta isolated from rats fed with control or HD, in comparison with non-stressed groups. CMS effects were independent of diet. In non-stressed rats, the HD induced dyslipidemia, but did not change glucose metabolism, vascular function, or morphology. The data from this study indicate that CMS promotes a set of events which together can contribute to impair function of the thoracic aorta.

Influence of different decalcifying agents on EGF and EGFR immunostaining

This study was designed to verify the influence of three demineralizing agents on EGF and EGFR immunostaining as well as on tissue morphology. We chose submandibular glands that are a source of EGF and its receptor and which could be analyzed using a control in which the decalcification step was not carried out. After sacrifice of adult male Wistar rats by perfusion fixation, the submandibular glands and mandibles were excised and placed together in each of the following solutions: (a) 5% nitric acid in 4% formaldehyde; (b) 4.13% EDTA pH 7.4; (c) 5% trichloroacetic acid. Mandibles served as a parameter for decalcification time in each demineralizing solution. A control group was performed with submandibular glands that were not placed in any demineralizing solution. After mandibles were completely decalcified, glands were processed by embedding in Paraplast® and immunohistochemical staining was made to detect EGF and EGFR. It was observed that decalcification did not produce noticeable differences in terms of EGF and EGFR immunoreactivity, but had an effect on the quality of the morphology and staining. Our results indicate there is no problem performing immunostaining of EGF and EGFR in tissues that require decalcification. 4.13% EDTA (pH 7.4) is the best choice for decalcification in cases that are not urgent.

Liposomal-benzocaine gel formulation: correlation between in vitro assays and in vivo topical anesthesia in volunteers

The aim of the present study was to characterize a liposome-based benzocaine (BZC) formulation designed for topical use on the oral mucosa and to evaluate its in vitro retention and permeation using the Franz-type diffusion cells through pig esophagus mucosa. To predict the effectiveness of new designed formulations during preclinical studies, a correlation between in vitro assays and in vivo efficacy was performed. Liposomal BZC was characterized in terms of membrane/water partition coefficient, encapsulation efficiency, size, polydispersity, zeta potential, and morphology. Liposomal BZC (BL10) was incorporated into gel formulation and its performances were compared to plain BZC gel (B10) and the commercially available BZC gel (B20). BL10 and B10 presented higher flux and retention on pig esophagus mucosa with a shorter lag time, when compared to B20. BZC flux was strongly correlated with in vivo anesthetic efficacy, but not with topical anesthesia duration. The retention studies did not correlate with any of the in vivo efficacy parameters. Thus, in vitro permeation study can be useful to predict anesthetic efficacy during preclinical tests, because a correlation between flux and anesthetic efficacy was observed. Therefore, in vitro assays, followed by in vivo efficacy, are necessary to confirm anesthetic performance.

Alveolar bone remodelling pattern of the rat incisor under different functional conditions as shown by minocycline administration

Remodelling of the socket surrounding the continuously growing and erupting rat incisor was examined in teeth under normo, hyper and hypofunctional conditions. Cross-sections of the mandible were observed under fluorescence microscopy, where minocycline labelling evidenced bone remodelling. Animals had received minocycline (10 mg/day) during the experimental period. Control animals (from all three groups) received vehicle alone and samples from these animals were not fluorescent. Minocycline did not interfere with the eruption rates in any of the functional conditions studied. Normofunctional (impeded) incisors showed constant osteogenic activity in the alveolar bone facing the periodontal ligament in all regions of the incisor. Under hypofunctional (unimpeded) and hyperfunctional (impeded) conditions, osteogenesis in the region close to the alveolar crest was markedly increased in the mesial wall of the socket. The labial alveolar bone, facing the enamel-related periodontium, was almost entirely formed during the experimental period in all the groups, but in hyper and hypofunctional teeth the newly formed bone was thicker and contained a substantial amount formed before the experimental period. In the more apical regions of the socket no marked differences between the three functional conditions were found. The similar bone remodelling shown in hypo and hyperfunctional teeth might indicate that there are common factors causing this pattern. Consideration of possible factors appear to rule out the eruption rate, which is very different under these two functional conditions.

The effects of local trauma to the enamel-related periodontal tissues in the eruption of the rat incisor.

The periodontal tissues related to enamel (PTE) of the rat incisor comprise a connective tissue derived from the dental follicle and the enamel organ with its successive stages of development. Localized damage to these tissues in rat lower incisors was done surgically in three ways: with an endodontic file introduced into the labial periodontal space through either (i) its basal or (ii) its incisal extremities, or (iii) by the partial removal of the mandibular lower border, at the level of the molar teeth, together with the introduction of an endodontic file into the incisal part of that space. The lesions in the molar region of the PTE produced first a variable period of retarded eruption, and, depending upon their extent or degree were followed by a cessation of the eruptive movement and, in the majority of the operated teeth, a recovery of the normal eruption rate before the end of the experiment (17 weeks after surgery). Access to the PTE through the basal portion of the socket was erratic, but when the tissues were damaged produced similar effects. Effects on eruption of lesions produced through the alveolar crest were minimal or even absent. Localized injury to the periodontal ligament of either lower or upper incisors did not produce similar effects on tooth eruption. The dental follicle and the enamel organ of teeth of limited growth when their crown is completed are similar to the PTE in the molar region of continuously growing rodent incisors. In teeth of limited growth these tissues play an essential part in the intraosseous stage of eruption. The results here suggest that the PTE may also have a role in the supraosseous stage of eruption, which is continuous in teeth such as rat incisors due to the presence of a continuously functioning odontogenic organ.

Intermittent Parathyroid Hormone Administration Improves Periodontal Healing in Rats

BACKGROUND Intermittent administration of parathyroid hormone (PTH) promotes new bone formation in patients with osteoporosis and bone fractures. It was shown previously that PTH also reduces periodontitis-related bone loss. The aim of this study is to evaluate the effect of treatment with PTH on periodontal healing in rats. METHODS Fenestration defects were created at the buccal surface of the distal root of the mandibular first molars, and both periodontal ligament (PDL) and cementum were removed. Animals were then assigned to two groups (eight animals per group): group 1: control, placebo administration; and group 2: test, human PTH (hPTH) 1-34 administration at a concentration of 40 μg/kg. For both groups, the animals were injected every 2 days, and the animals were sacrificed at 14 and 21 days after surgery. Specimens were harvested and processed for routine decalcified histologic sections. The following parameters were assessed: 1) remaining bone defect extension (RBDE); 2) newly formed bone density (NFBD); 3) total callus area (TCA); 4) osteoclast number (ON) in the callus region; and 5) newly formed dental cementum-like tissue (NFC). Birefringence of root PDL reattachment was also evaluated. RESULTS Birefringence analysis showed root PDL reattachment for both groups 21 days after treatment. Intermittent hPTH 1-34 administration decreased RBDE (P <0.01) and increased NFBD (P <0.01), TCA (P <0.01), area of NFC (P <0.01), and ON in the callus region (P <0.01). CONCLUSION Within the limits of the present study, intermittent administration of hPTH 1-34 led to an enhanced periodontal healing process compared with non-treated animals.