Effects of obesity on the rat incisor enamel and dentine thickness, as well as on the hemimandible shape over generations.

Obesity has several effects on the general body metabolism. However, little is known about the impact of obesity on the growth and shape of mineralized tissues like mandibles and teeth, as well as if it effects are passed down from generation to next. Therefore, in this study, we aimed to evaluate, over nine generations using the consanguineous mating (inbreeding), the effect of the obesity condition produced by the reduction in the number of rats per litter during the lactation period on the hemimandible shape, dentine, and enamel of the rat incisor. Litters were reduced to two males and two females after birth, and were consanguinity mated in adulthood for nine generations. For all evaluations performed in this investigation, only males were used. The control group was formed by a non-consanguineous litter containing eight males. The parameters evaluated were food consumption, body weight, Lee Index, and bone density of the hemimandible bone. Incisor enamel and dentine thickness were also evaluated. The hemimandible shape was evaluated using geometric morphometry. The results show a significant and progressive increase in food intake, Lee Index, body weight, hemimandible weight, and enamel thickness, and a decrease in dentine thickness. The linear measurements of the length of the ramus ascending hemimandibular segment were found to be shorter, while its height was increased. In contrast, the geometric morphometry shows that the general hemimandible shape changed over the consanguineous obesity generations. We conclude that over generations, obesity increases and maintains the parameters evaluated with significant changes in hemimandible shape as well as in the dimensions of enamel and dentine of incisors, suggesting that enamel and dentine could be used as phenotype biomarkers to detect changes in tooth and craniofacial development related to obesity effects.

Initial damage produced by a single 15-Gy x-ray irradiation to the rat calvaria skin.

BACKGROUND: Calvaria skin has a reduced thickness, and its initial damage produced by irradiation was scarcely reported. We aimed to identify the initial effects of x-ray irradiation in the rat calvaria skin. METHODS: After approval by the Animal Ethical Committee, calvaria skin sections of five Wistar rats per time point were evaluated on days 4, 9, 14, and 25 following a single 15-Gy x-ray irradiation of the head. The control group was composed of five rats and evaluated on day 4. Sections were assessed using hematoxylin-eosin and Masson's trichrome staining for morphology, inflammation, and fibrosis. Fibrosis was also evaluated by the collagen maturation index from Picrosirius red staining and by cell proliferation using the immunohistochemistry, after 5-bromo-2-deoxyuridine intraperitoneal injection. RESULTS: In irradiated rats, we observed a reduction in epithelial cell proliferation (p = 0.004) and in matrix metalloproteinase-9 expression (p < 0.001), an increase in the maturation index, and with a predominance in the type I collagen fibers, on days 9 and 14 (1.19 and 1.17, respectively). A progressive disorganization in the morphology of the collagen fibers at all time points and changes in morphology of the sebaceous gland cells and hair follicle were present until day 14. CONCLUSIONS: The initial damage produced by a single 15-Gy x-ray irradiation to the rat calvaria skin was a change in the normal morphology of collagen fibers to an amorphous aspect, a temporary absence of the sebaceous gland and hair follicles, and without a visible inflammatory process, cell proliferation, or fibrosis process in the dermis.

Goblet cells and intestinal Alkaline phosphatase expression (IAP) during the development of the rat small intestine.

This study aimed to evaluate the temporal and spacial distribution of the mucins produced by goblet cells and intestinal alkaline phosphatase (IAP) expression during the development of the small intestine of the rat. Intestines were removed from rats on the 15th, 17th and 18th days of intratuterine life (i.u.) and on the 3rd, 10th, 17th and 25th days after birth (a.b.). Intestines were processed for routine histological procedures and sections were submitted to histochemistry using PAS to stain neutral glycoproteins and Alcian blue for acidic glycoproteins, as well as immunohistochemistry to detect IAP. In rats, glycoprotein production was seen to begin in the intestinal epithelium cell at around the 17th day of i.u. life; however, this production was not accompanied by morphological indications of the presence of goblet cells. By the 18th i.u. day, the villus epithelium was undergoing differentiation and the first goblet cells could be identified from this time. At around the 10th day a.b., both compartments of the small intestine were detected; i.e. the villi and the crypts. At this timepoint, goblet cells were present in the villi, and also in the upper regions of the crypts. On the 3rd, 10th 17th and 25th days a.b., the presence of the goblet cells increased and presented regional differences in the sections evaluated. IAP was not detected during i.u. life, but was weakly detected in the cells of the villi from the 3rd day a.b., along the entire extension of the villi. On the 10th day, IAP was detected at the tip of the villi, while on the 25th day, it was detected along the extension of the villi, but with a weaker intensity. In conclusion, a temporal and spacial distribution of goblet cells and IAP activity occurs during the development of the small intestine, suggesting a possible regulatory control in accordance with the suckling and weaning phases of food intake in the rat's life.

Evaluation of the osteogenic potential of Hancornia speciosa latex in rat calvaria and its phytochemical profile.

ETHNOPHARMACOLOGICAL RELEVANCE: Hancornia speciosa Gomes, commonly known as Mangabeira, is a Brazilian native fruit tree belonging to the Apocynaceae family. In folk medicine, the latex obtained from Mangabeira's trunk has been used as an adjunct therapy for bone fractures. Few pharmacological studies on the Hancornia speciosa latex have been developed and despite its popular use for bone healing there is no data about its biological effect on bone. AIM OF THE STUDY: The present study aimed to investigate the osteogenic potential of Hancornia speciosa latex in rat calvaria, as well as its phytochemical profile. MATERIALS AND METHODS: A neutral gel composition containing 5% latex was topical applied to a critical size bone defect and over intact calvaria of rats. Areas of newly formed bone on the borders of the defect and of calvaria periosteum were quantified, as well as the percentage of BrdU-positive cells and total cells in the periosteum at different periods of time after latex application. The cytotoxicity of the latex aqueous phase was evaluated in rat calvarial cells in vitro by MTT assay and its phytochemical profile was investigated by ESI-MS/MS. RESULTS: The area of newly formed bone on the borders of the calvaria defect was larger in rats that received latex at 15 and 30 days of healing. After 3 days of latex application over the intact calvaria, the periosteum area was increased and newly formed bone was observed after 5 and 11 days. There was also an increase in periosteum cell proliferation and population followed latex application on calvaria (p<0.05). The latex aqueous phase limited rat calvarial cell viability in vitro in concentrations larger than 0.6mg/mL. Chlorogenic acid and naringenin-7-O-glucoside were identified in the latex aqueous phase, along with catechin and procyanidin compounds. CONCLUSION: There was a stimulus for periosteum cell proliferation and bone formation when Hancornia speciosa latex was topically applied on rat calvaria. In addition, chlorogenic acid and naringenin-7-O-glucoside present in Hancornia speciosa latex may contribute to its effects on bone formation.

MT1-MMP expression in the odontogenic region of rat incisors undergoing interrupted eruption.

MT1-MMP (membrane type matrix metalloproteinase-1) has been considered an important membrane-type matrix metalloproteinase involved in the remodeling process in tissue and organ development, including the processes of the tooth and root growth and dental eruption. Therefore, the aims of this study were to evaluate MT1-MMP expression in the odontogenic region, as well as the eruption rate and morphology of the lower-left rat incisor, where the eruption process was interrupted for 14 days by a steel wire attached from the center of the incisor labial face and braced to the first molar. In the interrupted eruption group, the eruption rate was significantly reduced, producing drastic morphological alterations in the tooth germ and socket area. The MT1-MMP expression was widespread in the dental follicle, in both groups studied (normal and interrupted eruption groups); however a significant decrease in immunostaining was observed in the interrupted eruption group. Results indicate that MT1-MMP may have an important role in the process of dental eruption.

Immunolocalization and activity of the MMP-9 and MMP-2 in odontogenic region of the rat incisor tooth after post shortening procedure.

MMP-9 and MMP-2 are metalloproteinases which degrade the denatured collagen fibers. However, there is no report about roles of these MMPs in the odontogenic region of the adult rat incisor tooth under different eruption conditions. Male Wistar rats were divided in a normofunctional group (NF) in which their lower teeth remained in a normal eruption. In a hypofunctional group (HP) rats underwent shortening of their lower left incisor tooth every 2 days during 12 days. The eruption rate as well as the expression and activities of MMP-9 and MMP-2 were evaluated using imunohistochemistry and zymography. Although the shortening increased the eruption rate, no changes in the MMP-9 and MMP-2 were observed. We conclude that in adult rats, in opposite to development of tooth, the MMP-9 and MMP-2 present in the odontogenic region does not seem to play a direct role in the remodeling matrix, even after post-shortening procedures which to lead an acceleration of the eruption process in the incisor.

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.

Increase of MT1-MMP, TIMP-2 and Ki-67 proteins in the odontogenic region of the rat incisor post-shortening procedure.

MT1-MMP and TIMP-2 are well known for their roles in remodelling of extracellular matrix components. However, reports are emerging on the involvement of these molecules in cell kinetics. In the rat incisor tooth, a shortening treatment increases the eruption and cell proliferation rates. However, the role of MT1-MMP and TIMP-2 proteins in these processes is still to be evaluated. Male Wistar rats were divided in two groups. In the normofunctional group (NF) the lower teeth of the rats remained in a normal eruption process. In the hypofunctional group (HP) rats their lower left incisor tooth was shortened every 2 days during 12 days. The eruption rate was estimated during the shortening period and MT1-MMP, TIMP-2 and Ki-67 protein expression from the odontogenic region was measured after the treatment. In HP groups an increase in eruption rate, and in MT1-MMP/TIMP-2 and Ki-67 expression were observed. We conclude that there is a relationship between the increase in eruption rate, and in levels of MT1-MMP, TIMP-2 and Ki-67 in the HP group. This suggests that MT1-MMP and TIMP-2 may have some role in cell proliferation during the eruption of the rat incisor tooth.