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Thirteen female Wistar rats were divided into two groups: one treated with ethanol and the other of untreated. Four newborns from each mother were selected and weighed, measured, and evaluated for physical characteristics. From these neonates, chondrocytes were extracted from the articular cartilages of the femur and tibia, and cultivated in a chondrogenic medium at 37oC and 5% CO2. At 7, 14, and 21 days of cultivation, alkaline phosphatase activity tests, MTT conversion to formazan, and percentage area covered by cells per field were performed. At 21 days, the percentage of PAS+ areas in 3D cultures was performed, as well as the evaluation of gene transcript expression for aggrecan, SOX-9, collagen type II, collagen X, Runx-2, and VEGF by real-time RT-PCR. The means were compared by Student’s t-test. The weight of the ethanol group neonates was significantly lower than that of the controls. Chondrocyte cultures from the ethanol group showed significantly higher AP activity, MTT conversion, and cell percentage. There was higher expression of collagen type II and lower expression of SOX-9 in the ethanol group. There was no difference in the percentage of PAS+ areas in pellets and in expression of aggrecan, collagen X, Runx-2, or VEGF between groups. In conclusion, prenatal exposure to ethanol alters the phenotype and activity of offspring chondrocytes, which may be mechanisms by which endochondral bone formation is compromised by maternal ethanol consumption.

This study aimed to evaluate the effect of maternal ethanol consumption during gestation and lactation on bone mass and osteogenic differentiation of mesenchymal stem cells of the bone marrow (BMMSCs) in rats. Thirteen adult Wistar rats were used. The rats were mated, and after confirmation of gestation, (day 0) they were distributed in two groups: the control group and the ethanol-treated group. From the ninth day of gestation, the rats of the ethanol and control groups were administered 40% alcoholic solution (4 g ethanol/kg) and distilled water, respectively, daily via gavage until the thirtieth day of lactation. The BMMSCs were extracted from the right femurs and tibiae and cultured using an osteogenic medium for 7, 14, and 21 days. The conversion of MTT to formazan crystals, alkaline phosphatase activity, and percentages of cells per field were analyzed. The number of mineralized nodules per field was examined, and quantification of the gene transcripts for osteopontin, osteocalcin, and BMP-2 was evaluated on day 21 by real-time RT-PCR. Morphometric evaluations of the percentage of trabecular bone and cortical thickness in the left femur and tibia were performed. The means were compared by the Student's t-test, and the differences were considered significant if p 0.05. The BMMSCs of the rats that consumed ethanol during gestation and lactation, when subjected to osteogenic differentiation in vitro, demonstrated higher conversion of MTT to formazan, higher alkaline phosphatase activity, a higher percentage of cells per field, higher expression of BMP-2, and higher synthesis of mineralized nodules when compared to those of control rat cells. However, there was no significant difference in the percentage of trabecular bone or cortical thickness between both groups. Hence, the consumption of ethanol during pregnancy and lactation did not alter the trabecular and cortical bone tissues of the femur and tibia compared with that of pregnant and lactating control rats that did not consume alcohol, despite BMMSCs showing higher osteogenic differentiation under in vitro conditions.

Summary Histopathological changes in tooth structures in dogs with calculus have not been described. The aim of this study was to evaluate the frequency of various histopathological changes in the dentine of teeth that had been surgically extracted from dogs with calculus. Data including breed, sex, age, reason for tooth extraction and dental history were obtained for each animal. A total of 158 teeth (45 incisors, 31 canines, 35 premolars and 47 molars) with calculus were extracted from 74 dogs of various ages and breeds. The teeth were decalcified, processed in paraffin wax and stained with haematoxylin and eosin for histopathological analysis. Of the 158 analysed teeth, 71 had dentinal changes, including 45 with external resorption cavities, 11 with osteodentine, six with internal resorption cavities, four with tertiary dentine, four with dentinal degeneration or fragmentation, and one with predentine degeneration or fragmentation. Canine teeth were the least commonly affected. Areas of dentinal degeneration or fragmentation unrelated to resorption cavities were only seen in the incisor and molar teeth. Dentinal changes and their frequencies were similar among the incisor, premolar and molar teeth. The presence or extension of dental calculus was not associated with the type or frequency of dentinal changes.

In forensic toxicology, the detection of toxic chemicals from human bone marrow is often used in cases with an extended post mortem interval; however, in veterinary medicine, this practice is not used. Therefore, this study was performed to investigate the suitability of bone marrow for toxicological analysis in dogs and cats. Six animals with suspected poisoning were selected; the carcasses were sent for necropsy, and the organs were collected and preserved in buffered formalin and processed routinely for histological examination. In addition, bone marrow samples from the femur, humerus, and tibia were collected for toxicological analysis by liquid chromatography coupled to mass spectrometry detection (LC-MS). This analysis confirmed the presence of aldicarb, aldicarb sulfone, asulam, carbendazim, chlorpyrifos, dichlorvos, thifensulfuron methyl and trifloxysulfuron-sodium and associated with clinical symptoms and anatomo-histopathological alterations it was recognized the poisonings. It is expected that this study will promote the toxicological investigation of bone marrow and open avenues for the use of this tissue as an option for the detection of toxic chemicals in cases of forensic pathology. RESUMO: Na toxicologia forense, a detecção de substâncias químicas tóxicas provenientes da medula óssea humana é frequentemente usada em casos com intervalo post mortem prolongado; no entanto, na medicina veterinária, essa prática não é utilizada. Portanto, este estudo foi realizado para investigar a utilização da medula óssea nas análises toxicológicas em cães e gatos. Seis animais com suspeita de intoxicação foram selecionados; as carcaças foram enviadas para necropsia e os órgãos foram coletados e preservados em formalina tamponada e processados rotineiramente para exame histológico. Amostras de medula óssea de fêmur, úmero e tíbia foram coletadas para análise toxicológica por cromatografia líquida acoplada à espectrometria de massa-massa (LC-MS). A análise por LC-MS confirmou a presença dos agrotóxicos aldicarbe, aldicarbe sulfona, asulam, carbendazim, clorpirifós, diclorvós, tifensulfuron metil e trifloxisulfuron-sódico, e em associação com sinais clínicos e achados anatomo-histopatológicos comprovou-se as intoxicações. Espera-se que este estudo promova a utilização da medula óssea como uma opção na investigação toxicológica para a detecção de produtos químicos tóxicos em casos de patologia forense.

The Solanum glaucophyllum Desf. has been used to treat and prevent diseases in human and veterinary medicine. On the other hand, plant poisoning causes several bone diseases, among them osteoporosis, which is characterized by osteoblastic hypoplasia. Because the osteoblast is a cell derived from the differentiation of mesenchymal stem cells (MSCs) from bone marrow, the hypothesis is that the plant reduces the osteogenic differentiation of MSCs. The objective of this study was to evaluate the effects of S. glaucophyllum Desf. extract on MSCs cultured in osteogenic differentiation medium. We determined by liquid chromatography that 1 ml of plant extract contained 3.8 μl of 1,25(OH)2 D3 (calcitriol). Four groups of MSCs cultivated in osteogenic medium were evaluated as follows: (a) treated with 100 μl of extract/L containing 0.4 μg/L of calcitriol; (b) treated with 1 ml of extract/L containing 4 μg/L of calcitriol; (c) treated with 5 ml of extract/L containing 20 μg/L of calcitriol; and (d) a control group without extract. We performed alkaline phosphatase activity assay, analysis of MTT conversion to formazan, and evaluated the percentage of cells, and number and diameter of mineralization nodules. The expression of gene transcripts for osteopontin, bone sialoprotein and BMP-2 was analysed by RT-qPCR. After 21 days, there was a significant reduction in MTT conversion to formazan in treated groups, of the cellularity in the group with 5 ml of extract/L, and in the number and size of mineralization nodules in the groups treated with 1 and 5 ml of extract/L. The 5 ml extract/L concentration also reduced transcript expression of osteopontin. It is concluded that S. glaucophyllum Desf. at concentrations of 1 and 5 ml extract/L reduced mineralized matrix synthesis in MSCs cultivated in osteogenic differentiation medium, which suggests that this is one of the mechanisms by which osteoporosis occurs in intoxicated animals.

Triple-negative tumors are characterized immunohistochemically by the absence of positivity to sex hormone receptors and to human epidermal growth factor receptor 2. Additionally, they are differentiated into basal-like and non-basal (or null) subtypes, based on the presence of basal cytokeratin expression (CK5/6, 14, and17). Triple-negative subtypes are yet to be characterized in male dogs, to our knowledge. We report herein the clinical and pathologic findings and molecular characterization of carcinoma in the mammary glands of 2 male dogs. Case 1 was diagnosed as a grade II tubulopapillary carcinoma; case 2 was diagnosed as a grade II carcinoma in a mixed tumor. The tumors were characterized phenotypically as triple-negative basal and triple-negative non-basal, respectively.

Objective We sought to evaluate the effect of different concentrations of ethanol on phenotype and activity of articular chondrocyte synthesis of neonatal rats in 2-dimensional (2D) and 3-dimensional (3D) culture. Methods Chondrocytes were cultured in chondrogenic medium with different concentrations of ethanol: 0.0% v/v (control); 0.05% v/v (8.6 mM); 0.25% v/v (42.9 mM), and 0.5% v/v (85.7 mM). Chondrocytes under 2D culture were subjected to MTT assay, while chondrocytes under 3D culture were processed for paraffin inclusion and stained by periodic acid Schiff (PAS) to evaluate mean chondrocyte diameter and percentages of cells, nucleus, cytoplasm, well-differentiated matrix, and PAS+ areas. The expression of gene transcripts for aggrecan, Sox9, and type II collagen was evaluated by real-time quantitative polymerase chain reaction. Results There was no difference between groups by the MTT assay. PAS staining revealed that chondrocytes treated with 0.5% v/v ethanol had higher percentages of cytoplasm and nuclear areas, but with a reduction in PAS+ matrix area. The mean diameter of chondrocytes was similar between groups. The expression of aggrecan in the group treated with 0.5% v/v ethanol was lower in comparison to that in the control. In the groups treated with 0.25% v/v and 0.5% v/v ethanol, the percentage of differentiated cartilage was lower in comparison with that in the control. The group treated with 0.05% v/v ethanol was similar to the control in all parameters. Conclusions Ethanol acted directly on in vitro cultured articular chondrocytes of newborn rats, altering the chondrocyte phenotype and its synthesis activity, and these effects were dose dependent.

The Kisspeptin/Kiss1r system is a key regulator of reproduction by stimulating gonadotrophinreleasing hormone and luteinizing hormone release, and in vitro studies have shown that Kisspeptin can modulate angiogenesis and immune function, factors that are also essential for reproduction However, there are no studies on the expression of Kisspeptin/Kiss1r at the maternal-fetal interface in domestic cats and its relationship with angiogenic and immunological mediators. Thus, our objective was to evaluate the spatiotemporal expression profile of Kisspeptin/Kiss1r and angiogenic and immunological mediators in the uterus and placenta of domestic cats during pregnancy. Uterus and placenta samples were collected from cats in mid pregnancy (N = 6) and late pregnancy (N = 6), in addition to uterus from non-pregnant cats in diestrus (N = 7), to evaluate protein and gene expression of kisspeptin (Kiss1), kisspeptin receptor (Kiss1r), vascular endothelial growth factor (VEGF), tyrosine kinase receptor (Flk-1), placental growth factor (PLGF), interferon gamma (INFγ), migration inhibiting factor (MIF), tumor necrosis factor (TNFα), interleukins (IL6 and IL10) by immunohistochemistry and quantitative polymerase chain reaction. Pregnancy increased the uterine expression of Kiss1 and Kiss1r, especially at the late pregnancy, in addition to upregulating INFy, MIF, Vegf, Il10, and Tnf and downregulating Plgf. Higher placental expression of Kiss1r and Plgf mRNA occurred at the late pregnancy, while the expression of Kiss1, VEGF, Flk-1, INFy, TNFα, Il6, and IL10 was higher in the mid of pregnancy. A positive correlation between Kiss1 and Tnf was observed in the placenta, while Kiss1r had a negative correlation with Infγ, Il6, and Il10. The findings reveal that Kisspeptin/Kiss1r and angiogenic and immunological mediators at the maternal-fetal interface of pregnant cat have a gene correlation and are modulated by the gestational age. These data suggest possible functional links of Kisspeptin in placental angiogenesis and immunology.Summary sentencePregnancy in cats stimulates the uterine expression of the Kiss1/Kiss1r system, especially in late pregnancy, and Kiss1/Kiss1r and angiogenic and immune factors at the cat's placenta have a gene correlation and are modulated by the gestational age.

Failures in hypothalamic kisspeptin/Kiss1r signaling are associated with infertility, and in vitro studies have shown that kisspeptin can modulate angiogenesis and immune activity. Because there is no in vivo research on the functional relationship between these factors in the reproductive system, especially in domestic cats, we evaluated the expression profile of kisspeptin/Kiss1r and angiogenic and immunological mediators in the genital tract of cyclic cats and of those with pyometra. The uterus of cats in diestrus exhibited greater gene and protein expression of Kiss1, as well as Vegf, Pigf, Mif, and Il6. In contrast, Kiss1r presented greater expression in proestrus/estrus, similarly to that observed for the immunostaining of INFγ, MIF, TNFα, and IL10. These factors were positively correlated with Kiss1 and/or Kiss1r, and a positive correlation between Kiss1 and Kiss1r was also observed in the uterus of cats during the estrous cycle. Cats with pyometra showed greater immunostaining of Kiss1 and Kiss1r on the endometrial surface and reduced immunostaining of Kiss1 in deep glands, whereas there was a significant reduction in Vegf, Pigf, Mif, and Il6 mRNA, and an increase in Tnf mRNA. The findings reveal that there is a gene correlation between kisspeptin/Kiss1r and angiogenic and immune mediators in the uterus of the domestic cat, which is modulated by the estrous cycle, and that pyometra affects the expression of these mediators. This study suggests, for the first time, a functional relationship between the Kiss/Kiss1r system and angiogenic and immune mediators in the female genital tract.

The use of stem cells in cell therapies has shown promising results in the treatment of several diseases, including diabetes mellitus, in both humans and animals. Mesenchymal stem cells (MSCs) can be isolated from various locations, including bone marrow, adipose tissues, synovia, muscles, dental pulp, umbilical cords, and the placenta. In vitro, by manipulating the composition of the culture medium or transfection, MSCs can differentiate into several cell lineages, including insulin-producing cells (IPCs). Unlike osteogenic, chondrogenic, and adipogenic differentiation, for which the culture medium and time are similar between studies, studies involving the induction of MSC differentiation in IPCs differ greatly. This divergence is usually evident in relation to the differentiation technique used, the composition of the culture medium, the cultivation time, which can vary from a few hours to several months, and the number of steps to complete differentiation. However, although there is no “gold standard” differentiation medium composition, most prominent studies mention the use of nicotinamide, exedin-4, ß-mercaptoethanol, fibroblast growth factor b (FGFb), and glucose in the culture medium to promote the differentiation of MSCs into IPCs. Therefore, the purpose of this review is to investigate the stages of MSC differentiation into IPCs both in vivo and in vitro, as well as address differentiation techniques and molecular actions and mechanisms by which some substances, such as nicotinamide, exedin-4, ß-mercaptoethanol, FGFb, and glucose, participate in the differentiation process.

This study aimed to evaluate the effects of thyroid hormone on the decidua and metrial gland of rats and to examine the expression of angiogenic factors. 72 adult, female rats were divided into hypothyroid, T4-treated2, and control groups. At 10, 14 and 19 days of gestation (DG), the decidua and metrial gland were collected for histomorphometric and immunohistochemical evaluation of the expression of VEGF, Flk-1 and Tie-2. Hypothyroidism reduced the area of the decidua at 10 and 19 DG. Furthermore, VEGF was increased at 10 and 14 DG, and Flk-1 only at 14 DG, but both was reduced at 19 DG in the metrial gland without significantly changing the area occupied by blood vessels. Rats treated with T4 showed an increase in the decidua blood vessels at 10 and 19 DG. However, at 10 DG, excess T4 resulted in increased of Flk-1 in the decidua and metrial gland. Hypothyroidism increased the Tie-2 at 10 and 19 DG in the decidua and metrial gland. In conclusion, hypothyroidism reduces the area of the decidua and increases the expression of VEGF, Tie-2 and Flk-1. The excess of T4 promotes tissue angiogenesis by increasing the number of vessels in the decidua because of the increased expression of Flk-1.

The hypothesis of this experiment is that mesenchymal stem cells (MSCs) are involved in the genesis of the bone metaplasia caused by Solanum glaucophyllum intoxication. We determined using liquid chromatography that 1 mL of plant extract contained 3.8 μl of 1,25(OH)2D3. The ability of 100 μL, 1 mL and 5 mL of extract/L, containing 1 nM (0.4 μg/L), 10 nM (4 μg/L) and 50 nM (20 μg/L) of 1,25(OH)2D3, respectively, in inducing the osteogenic differentiation in bone marrow MSCs from rats was tested. At the concentrations of 1 and 5 mL of extract/L of culture medium without osteogenesis-inducing factors, the plant extract induced the osteogenic differentiation of the MSCs, as was evidenced by the greater synthesis of mineralized matrix. At the higher concentration (5 mL of extract/L), an increase in the relative expression of BMP-2 gene was observed. It was concluded that rat bone marrow MSC culture is a good model for studying the effects of the S. glaucophyllum extract on the osteogenic differentiation of undifferentiated cells. Also, S. glaucophyllum extracts containing 10 nM (4 μg/L) and 50 nM (20 μg/L) of 1,25(OH)2D3 induce the osteogenic differentiation of MSCs, suggesting that this is one of the mechanisms by which S. glaucophyllum causes bone metaplasia.

Caffeine is an alkaloid that is widely consumed due to its presence in drugs, coffee, tea, and chocolate. This compound passes to offspring through the placenta and milk; can cause teratogenic mutations; and reduces the formation, growth, and mass of bone. Because mesenchymal stem cells (MSCs) are responsible for generating the entire skeleton, we hypothesized that these cells are targets of caffeine. This study evaluated the osteogenic differentiation of MSCs derived from the offspring of rats treated with caffeine during pregnancy and lactation. Twenty-four adult Wistar rats were randomly divided into four groups, including one control group and three experimental groups treated with 25, 50, or 100 mg/kg of caffeine. At weaning, three 21-day-old pups from each dam in each group were euthanized for extraction of bone marrow cells for in vitro tests. Caffeine doses of 50 and 100 mg/kg significantly reduced the activity of alkaline phosphatase at 7, 14, and 21 days and the expression of collagen I at 21 days. However, the expression of gene transcripts for alkaline phosphatase, Runx-2, and bone sialoprotein, as well as the synthesis of mineralization nodules, decreased significantly in all groups treated with caffeine. The expression of osteocalcin was significantly reduced only in the group treated with 50 mg/kg caffeine. The caffeine that passes from the mother to the offspring during pregnancy and lactation reduces the osteogenic differentiation of MSCs. We propose that this reduction in the osteogenic potential of MSCs may be involved in the pathogenesis of osteopenia resulting from caffeine consumption.

Osteoporosis is a metabolic disease that affects bone tissue and is highly associated with bone fractures. Typical osteoporosis fracture treatments, such as bisphosphonates and hormone replacement, present important challenges because of their low bioavailability on the site of action. Options to overcome this issue are systems for the local release of therapeutic agents such as bioactive glasses containing therapeutic molecules and ions. These agents are released during the dissolution process, combining the drugs and ion therapeutic effects for osteoporosis treatment. Among the therapeutic agents that can be applied for bone repair are strontium (Sr) ion and phytopharmaceutical icariin, which have shown potential to promote healthy bone marrow stem cells osteogenic differentiation, increase bone formation and prevent bone loss. Submicron Sr-containing bioactive glass mesoporous spheres with sustained ion release capacity were obtained. Icariin was successfully incorporated into the particles, and the glass composition influenced the icariin incorporation efficiency and release rates. In this work, for the first time, Sr and icariin were incorporated into bioactive glass submicron mesoporous spheres and the in vitro effects of the therapeutic agents release were evaluated on the reduced osteogenic potential of rat osteoporotic bone marrow mesenchymal stem cells, and results showed an improvement on the reduced differentiation potential.

EnglishThe aim of this study was to evaluate the use of mesenchymal stem cells (MSC) in the treatment of myonecrosis induced by Bothrops alternatus venom in rats. Seventy-five male adult Wistar rats were divided into three experimental groups. G1 and G2 were injected in the gastrocnemius muscle with 120μg of B. alternatus venom, while G3 received 200μL of PBS only. Three days after the venom injection, 12 rats from G1 were treated with 5.0 x 106 MSC in PBS, whereas G2 and G3 rats received PBS. Every three days, blood and muscle samples of five animals from each group were taken for serum biochemical and pathological analyses. Histological examinations showed more intense muscle lesions following MSC treatment, characterized by disorganization and loss of muscle fibers, with focal necrosis and inflammatory infiltration by mononuclear cells. In conclusion, the use of MSC for the treatment of local damage caused by inoculation of B. alternatus venom impaired muscle regeneration and interfered in the healing process. Key words: Viperidae envenomation; stem cells; rats; myonecrosis; muscular repair portuguesO objetivo do presente trabalho foi avaliar a utilizacao das celulas tronco mesenquimais (MSC) no tratamento da mionecrose induzida pelo veneno de Bothrops alternatus em ratos. 75 ratos Wistar adultos foram distribuidos em tres grupos experimentais. G1 e G2 receberam 120μg de veneno de B. alternatus, enquanto o G3 recebeu apenas 200μL de PBS. Tres dias apos a administracao do veneno, os ratos do grupo G1 foram tratados com 5.0 x 106 MSC, enquanto G2 e G3 receberam exclusivamente PBS. A cada tres dias, amostras de sangue e tecido muscular foram coletadas de cinco animais de cada grupo para avaliacao bioquimica serica e patologica, respectivamente. A analise histologica revelou lesao muscular mais intensa apos a aplicacao das MSC, caracterizada pela desorganizacao e perdas das fibras musculares, com necrose focal e infiltrado inflamatorio mononuclear. E possivel concluir que a utilizacao das MSC para o tratamento da lesao local causada pela inoculacao do veneno B. alternatus deteriorou a regeneracao muscular e interferiu com o processo de cicatrizacao. Palavras-chave: envenenamento por cobra; celulas tronco; ratos; mionecrose; reparacao muscular

Objective Evaluate the effects of doses of caffeine administered to pregnant rats on the articular cartilage chondrocytes of their offspring. Methods Twenty-four adult Wistar rats were randomly assigned to four groups, with one control group and three groups being treated with caffeine at doses of 25, 50 and 100 mg/kg throughout pregnancy. At birth, three offspring/females were euthanized so that the chondrocytes could be extracted. At 7, 14 and 21 days of culture, the chondrocytes were subjected to the MTT cell viability assay and an evaluation of their alkaline phosphatase activity and collagen synthesis. Chondrocytes were also stained by Hematoxylin-eosin, PAS, Safranin-O and Alcian Blue. The Sox-9, Runx-2, aggrecan, collagen-II and alkaline phosphatase gene transcript levels were also evaluated. Mean comparisons were performed by the Student–Newman–Keuls test. Results Chondrocyte cultures from the 25 mg/kg group had the lowest results, as chondrocytes from this group had reduced viability, percentage of cells, alkaline phosphatase activity and collagen and chondrogenic matrix synthesis. A reduced expression of Sox-9, alkaline phosphatase and collagen-II was also detected in the 25 mg/kg group. Chondrocyte cultures of the group treated with 50 mg/kg caffeine showed reduced collagen synthesis and Sox-9 expression. The caffeine dose of 100 mg/kg also reduced collagen and Sox-9 and alkaline phosphatase expression. Conclusion Caffeine administered to pregnant rats negatively alters the articular cartilage chondrocytes of their offspring, reducing the synthesis of collagen and Sox-9 expression regardless of the dose. This study also concluded that the effects of caffeine are not linear or dose-dependent.

New Findings What is the central question of this study? Clinical studies suggest that obesity ‘protects’ against osteoporosis. However, these studies used only bone densitometry and assessed only one bone site, which is insufficient to enable conclusions to be drawn about the response of the whole skeleton. Furthermore, the effects of exercise on bone responses in obesity have not been explored previously. What is the main finding and what is its importance? We show that obesity causes osteopetrosis. Therefore, the classical perspective of ‘protective effects of obesity’ needs to be reviewed, and exercise is an important tool to avoid these alterations and to maintain the homeostasis of bone. A sedentary lifestyle and obesity induce systemic inflammatory responses. Although the effects of physical inactivity on osseous tissue have been well established, the effects of obesity on bone tissue remain controversial. Furthermore, the effects of physical training on bone tissue responses in the presence of diet-induced obesity are unknown. Our aim was to investigate the effects of obesity and physical training at multiple bone sites in rats. Female Wistar rats were divided into the following four groups: (i) control diet, non-trained (C-NT); (ii) high-refined carbohydrate-containing diet, non-trained (HC-NT); (iii) control diet, trained (C-T); and (iv) high-refined carbohydrate-containing diet, trained (HC-T). At 5 months of age, the rats were submitted to daily exercise for 30 min day−1. After 13 weeks, blood samples, adipose and skeletal tissues were harvested. Two-way ANOVA was applied to detect differences (significance accepted when P ≤ 0.05). The HC-NT group exhibited increased body mass, adiposity, serum leptin, serum insulin, insulin resistance index and concentrations of tumour necrosis factor-α and interleukin-6. Obese rats (HC-NT) exhibited thickening of nasal bones, trabecular bones in the lumbar vertebrae and long bones in a site-dependent manner. The HC-T group exhibited similar adiposity and inflammatory results. Morphological analysis of the lumbar vertebrae in rats fed the HC diet revealed characteristics of osteopetrosis that were inhibited by exercise. In conclusion, the HC diet induced obesity and inflammatory/hormonal alterations and increased the trabecular bone in a site-dependent manner. However, obesity caused osteopetrosis in the lumbar vertebrae, which could be inhibited by physical training. Although exercise inhibited the development of bone alterations, physical training did not inhibit the HC diet-induced obesity responses.

The objective of this study was to establish a protocol for the isolation and culture of mesenchymal stem cells (MSC) from bone marrow and adipose tissue of dogs. Three 6-month-old male dogs were used. Approximately 3.0 cm3 of adipose tissue and 3.0 mL of bone marrow were collected. The samples were processed and the isolated cells were cultured in DMEM. The cells were subjected to phenotypic characterization and to osteogenic, adipogenic, and condrogenic differentiation to confirm the isolation of the MSC. The cells showed elongated and fusiform morphology and they were able to differentiate into osteoblasts, adipocytes, and chondrocytes. Phenotypic characterization revealed high expression of the MSC markers CD90(80.04%) and CD29(96%) in the cells from bone marrow and high expression of CD90(60.94%) and CD29(77.08%) in the cells from adipose tissue. In addition, phenotypic characterization revealed low expression of hematopoietic markers CD45(1.45%) and CD34(1.53%) in the cells from bone marrow and low expression of CD45(1.45%) and CD34(1.53%) in the cells from adipose tissue. Based on these results, the modifications applied to classical protocols simplified the process and proved to be efficient in the isolation, culture, and expansion of MSC isolated from the bone marrow and adipose tissue of dogs. Keywords: cell culture; differentiation; regenerative medicine.

ObjectivesVerify the in-vitro effect of triiodothyronine (T3) on the chondrogenic differentiation of female rat bone marrow mesenchymal stem cells (BMMSCs) over several time periods and at several doses.MethodsCD54 + /CD73 + /CD90 + BMMSCs from Wistar female rats were cultured in chondrogenic medium with or without T3 (0.01; 1; 100; 1000 nm). At seven, 14 and 21 days, the cell morphology, chondrogenic matrix formation and expression of Sox9 and collagen II were evaluated.Key findingsThe dose of 100 nm did not alter the parameters evaluated in any of the periods studied. However, the 0.01 nm T3 dose improved the chondrogenic potential by increasing the chondrogenic matrix formation and expression of Sox9 and collagen II in at least one of the evaluated periods; the 1 nm T3 dose also improved the chondrogenic potential by increasing the chondrogenic matrix formation and the expression of collagen II in at least one of the evaluated periods. The 1000 nm T3 dose improved the chondrogenic potential by increasing the chondrogenic matrix formation and Sox9 expression in at least one of the evaluated periods.ConclusionsT3 has a dose-dependent effect on the differentiation of BMMSCs from female rats.

Purpose/Aim: The aim of this study was to evaluate the effects of excess maternal and postnatal thyroxine on chondrocytes and the extracellular matrix (ECM) of growth cartilage.We used 16 adult female Wistar rats divided into two groups: thyroxine treatment and control. From weaning to 40 days of age, offspring of the treated group (n = 8) received L-thyroxine. Plasma free T4 was measured. Histomorphometric analysis was performed on thyroids and femurs of all offspring. Alcian blue histochemical staining and real-time reverse transcription polymerase chain reaction measurements of gene expression levels of Sox9, Runx2, Aggrecan, Col I, Col II, Alkaline phosphatase, Mmp2, Mmp9, and Bmp2 were performed. Data were analyzed for statistical significance by student's t-test.Excess maternal and postnatal thyroxine reduced the intensity of Alcian blue staining, altered the number of chondrocytes in proliferative and hypertrophic zones in growth cartilage, and reduced the gene expression of Sox9, Mmp2, Mmp9, Col II, and Bmp2 in the growth cartilage of all offspring. Additionally, excess thyroxine altered the gene expression of Runx2, Aggrecan and Col I, and this effect was dependent on age.Excess thyroxine in neonates suppresses chondrocyte proliferation, stimulates chondrocyte hypertrophy and changes the ECM composition by reducing the amount of proteoglycans and glycosaminoglycans (GAGs). Prolonged exposure to excess thyroxine suppresses chondrocyte activity in general, with a severe reduction in the proteoglycan content of cartilage and the expression of gene transcripts essential for endochondral growth and characteristics of the chondrocyte phenotype.

The objective of the present study was to evaluate the gene and immunohistochemical expression of inflammatory mediators involved in the immune activity and the intrauterine trophoblast migration of the placentas in hypothyroid and L-thyroxine (L-T4)-treated rats. A total of 144 adult female rats were divided equally into hypothyroid,l-T4-treated, and euthyroid (control) groups. Hypothyroidism was induced by daily administration of propylthiouracil. Rats were killed at 0, 10, 14, 15, 16, 17, 18, and 19 days of gestation. We evaluated the depth of interstitial and endovascular intrauterine trophoblast invasion and the immunohistochemical expression of interferon γ (INFy), migration inhibitory factor (MIF), and inducible nitric oxide synthase (NOS2 (iNOS)). The gene expression of Toll-like receptor 2 (Tlr2) andTlr4,Infy,Mif, tumor necrosis factor (Tnf(Tnfα)),Il10,Nos2, matrix metalloproteinase 2 (Mmp2) andMmp9, and placental leptin was also measured in placental disks by real-time RT-PCR. The data were analyzed using an Student-Newman-Keuls (SNK) test. Hypothyroidism reduced the endovascular and interstitial trophoblast migration, and the expression of TLR4, INFy, MIF, interleukin 10 (IL10), NOS2, MMP2 and MMP9, and placental leptin, while increased the expression of TLR2 (P4-treated rats not only increased the expression of IL10 and NOS2 but also reduced the expression of TNF and MIF at 10 days of gestation (P4-treated group (P4also increased the gene expression ofMmp2at 10 days of gestation (PP4differentially affect the immune profile and the intrauterine trophoblast migration of the placenta, and these effects are dependent on the gestational period.

Osteoporosis causes reduction of osteogenic differentiation of mesenchymal stem cells (MSCs) from bone marrow and adipose tissue. This study was designed to compare the osteogenic potential of bone marrow mesenchymal stem cells (BMMSCs) and adipose-derived stem cells (ADSCs) of ovariectomized (OVX) rats. MSC were harvested from bone marrow and inguinal fat pads of six OVX rats. The limitations of this report are that cells from different animals were pooled for the purpose of the experiments that were carried out in this study. At 7, 14 and 21 d of osteogenic differentiation, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) conversion, alkaline phosphatase activity and gene expression for collagen I, osteocalcin, bone sialoprotein, osteopontin and bone morphogenetic protein-2 bone morphogenetic protein-2 (BMP-2) were analyzed. At 21 d, percentage of cells per field and percentage of mineralized nodule were analyzed. The data were subjected to analysis of variance, and the means were compared by Student-Newman-Keuls test. The cells, regardless of group, showed phenotypic characteristics consistent with stem cells. MTT conversion, alkaline phosphatase activity, percentage of mineralized nodule and expression of collagen I, osteocalcin and BMP-2 of ADSCs from OVX rats were higher when compared to BMMSCs from OVX rats in at least one of the evaluated periods (p

Objective The aim of this study was to unravel the mechanisms by which thyroxine affects skeletal growth by evaluating proliferative activity and angiogenic profile of growth cartilage of neonatal and weanling rats. Methods Sixteen adult Wistar rats were equally divided into 2 groups: control and treated with thyroxine during pregnancy and lactation. The weight, measurement of plasma free T4 and thyroids, femurs' histomorphometric analysis, and proliferative activity and angiogenic profile by immunohistochemical or real-time reverse transcriptase-polymerase chain reaction in growth cartilage was performed. Data were analyzed using Student's t test. Results The free T4 was significantly higher in the treated rats. However, the height of the follicular epithelium of the thyroid in newborns was significantly lower in the treated group. The excess maternal thyroxine significantly reduced the body weight and length of the femur in the offspring but significantly increased the thickness of trabecular bone and changed the height of the zones of the growth plate. Furthermore, excess maternal thyroxine reduced cell proliferation and vascular endothelial growth factor (VEGF) expression in the growth cartilage of newborn and 20-day-old rats ( P < 0.05). There was also a reduction in the immunohistochemical expression of Tie2 in the cartilaginous epiphysis of the newborns and FLK-1 in the articular cartilage of 20-day-old rats. No significant difference was observed in Ang2 expression. Conclusions The excess maternal thyroxine during pregnancy and lactation reduced endochondral bone growth in the progeny and reduced the proliferation rate and VEGF, Flk-1, and Tie2 expression in the cartilage of growing rats without altering the mRNA expression of Ang1 and Ang2.

OBJETIVO: Avaliar se a triiodotironina (T3) aumenta a diferenciação osteogênica das células-tronco mesenquimais do tecido adiposo (CTM-TA) de ratas adultas ovariectomizadas e com osteoporose e compará-lo ao de ratas adultas e jovens sem osteoporose. MATERIAIS E MÉTODOS: CTM-TA foram cultivadas em meio osteogênico e distribuídas em sete grupos: 1) CTM-TA de ratas jovens sem osteoporose; 2) CTM-TA de ratas adultas sem osteoporose; 3) CTM-TA de ratas adultas com osteoporose e 4, 5, 6 e 7) CTM-TA de ratas adultas com osteoporose tratadas com T3 (0,01 nM, 1 nM, 100 nM e 1.000 nM). AVALIARAM-SE: atividade da fosfatase alcalina, conversão do dimetiltiazol (MTT), porcentagem de nódulos de mineralização, celularidade e quantificação de transcriptos gênicos para colágeno I, osteocalcina, osteopontina e Bmp-2. RESULTADOS: Independente da dose, T3 reduziu a conversão do MTT, a atividade da fosfatase, a porcentagem de células e a expressão de colágeno I em pelo menos uma das doses e dos períodos estudados (p < 0,05). Mas o tratamento com T3 não alterou o número de nódulos de mineralização e a expressão de osteopontina e Bmp-2 em culturas de CTM-TA de ratas adultas com osteoporose (p > 0,05). CONCLUSÃO: T3 apresenta efeitos negativos sobre alguns fatores envolvidos na diferenciação osteogênica de CTM-TA, sem, no entanto, reduzir a formação de nódulos de mineralização e a expressão de proteínas ósseas. OBJECTIVE: To examine if triiodothyronine (T3) increases osteogenic differentiation adipose tissue derived stem cells (ASCs) from ovariectomized adult rats with osteoporosis compared with young rats and adult rats without osteoporosis. MATERIALS AND METHODS: The ASCs were cultured in osteogenic medium and distributed into seven groups: 1) ASCs of young rats without osteoporosis; 2) ASCs of adult rats without osteoporosis; 3) ASCs of adult rats with osteoporosis and 4, 5, 6 and 7) ASCs of adult rats with osteoporosis treated with T3 (0.01 nM, 1 nM, 100 nM and 1,000 nM). We analyzed alkaline phosphatase activity, dimethylthiazol (MTT) conversion, percentage of mineralized nodules, cellularity and quantification of gene transcripts for collagen I, osteocalcin, osteopontin and Bmp-2. RESULTS: Regardless of the dose, T3 reduced the MTT conversion, alkaline phosphatase activity, percentage of cells and the expression of collagen I in at least one of the doses and periods studied (p < 0.05). But, the treatment with T3 does not modify the number of mineralized nodules and the expression of osteopontin and Bmp-2 in culture of ASCs from adult rats with osteoporosis (p > 0.05). CONCLUSION: T3 has a negative effect on some factors involved in osteogenic differentiation of ASCs from adult rats with osteoporosis, without; however, reduce the formation of mineralized nodules and the expression of bone proteins.

Objective: To evaluate, in rat offspring, bone changes induced by excess maternal thyroxin during pregnancy and lactation, and to assess the reversibility of these changes after weaning. Material and methods: Twenty Wistar rats were distributed in two groups, hyperthyroid and control, that were treated daily with L-thyroxin (50 mcg/animal) and placebo, respectively. The treatment was initiated seven days before mating and continued throughout pregnancy and lactation. From every female of each of the two groups, two offspring were euthanized after birth, two at 21 days of age (weaning), and two at 42 days of age (21 days after weaning). In newborns, the length of pelvic and thoracic limbs were measured, and in the other animals, the length and width of the femur and humerus were measured. Bones were dissected, decalcified, embedded in paraffin, and analyzed histomorphometrically. Results: Excess maternal thyroxin significantly reduced the length of the pelvic limb in neonates. In 21-day-old individuals, excess maternal thyroxine reduced the length and the width of the femur and the humerus. It also increased thickness of the epiphyseal plate and the percentage of trabecular bone tissue. In 42-day-old individuals, there were no significant differences between groups in relation to the parameters evaluated in the previous periods. Conclusion: Excess maternal thyroxine reduced growth in suckling rats both at birth and at weaning, and it also increased the percentage of trabecular bone tissue in 21-day-old animals. These changes, however, were reversible at 42 days, i.e., 21 days after weaning. Objetivo: Avaliar, na prole de ratos, as alterações ósseas induzidas pelo excesso de tiroxina materna durante gravidez e lactação, e avaliar a reversibilidade dessas alterações após o desmame. Materiais e métodos: Vinte ratos Wistar foram distribuídos em dois grupos, hipertireoide e controle, que foram tratados diariamente com L-tiroxina (50 mcg/animal) e placebo, respectivamente. O tratamento foi iniciado sete dias antes do acasalamento e continuou durante toda a gravidez e lactação. De cada fêmea de cada um dos dois grupos, dois filhotes foram sacrificados após o nascimento, dois aos 21 dias de idade (desmame), e duas aos 42 dias de idade (21 dias após o desmame). Em recém-nascidos, o comprimento dos músculos pélvico e torácico membros foram medidos, e nos demais animais, o comprimento e a largura do fêmur e do úmero foram medidos. Os ossos foram dissecados, descalcificados, incluídos em parafina e analisados ​​histomorfometricamente. Resultados: O excesso de tiroxina materna reduziu significativamente o comprimento do membro pélvico em neonatos. Em indivíduos de 21 dias de idade, o excesso de tiroxina materna reduziu o comprimento e a largura do fêmur e do úmero. Também aumentou a espessura da placa epifisária e a porcentagem do tecido ósseo trabecular. Em indivíduos com 42 dias de idade, não houve diferenças significativas entre grupos em relação aos parâmetros avaliados nos períodos anteriores. Conclusão: Excesso materno A tiroxina reduziu o crescimento em ratas lactantes tanto ao nascimento quanto ao desmame, e também aumentou a porcentagem de tecido ósseo trabecular em animais com 21 dias de idade. Essas mudanças, no entanto, foram reversíveis em 42 dias, ou seja, 21 dias após o desmame.

The effect of intra-bone injection of differentiated rat bone marrow mesenchymal stem cells (BMMSCs) into the femur of osteoporotic female rats was studied. Osteoporosis was induced in Wistar female rats by bilateral ovariectomy. Then, 0.75 million BMMSCs isolated from healthy rats were injected into the femurs of osteoporotic rats. Histomorphometric analysis and histology clearly revealed improvements in the treated group as compared to untreated group. In 2 months, the femurs of treated rats, unlike untreated rats, showed trabecular bone percentage almost similar to the femurs from control healthy rats. To confirm the origin of newly formed bone, the experiment was repeated with BMMSCs isolated from green fluorescent protein transgenic rats. Confocal microscopy demonstrated green fluorescent protein-positive cells at the surface of trabecular bone of the treated rats. We investigated in vitro osteogenic differentiation of BMMSCs isolated from osteoporotic rats by studying alkaline phosphatase activity, collagen synthesis, and the ability to form mineralized nodules. Osteoporotic BMMSCs showed less differentiation capabilities as compared to those isolated from healthy rats. The results clearly demonstrated the importance of BMMSCs in osteoporosis and that the disease can be treated by injection of BMMSCs.

Bioactive glass/polymer hybrids are promising materials for biomedical applications because they combine the bioactivity of these bioceramics with the flexibility of polymers. In previous work hybrid foams with 50% bioactive glass and 50% polyvinyl alcohol (PVA) were prepared by the sol-gel process for application as scaffold for bone tissue engineering. In this work the hybrid samples were tested in osteoblast culture to evaluate adhesion and proliferation. Samples were also implanted subcutaneously in the dorsal region of adult rats. The hybrid 50% PVA/bioactive glass foam was chosen as the best scaffold in the composition range studied and it is a promising material for bone repair, providing a good environment for the adhesion and proliferation of osteoblasts in vitro. Concerning the in vivo studies we can assure that the “foreing body” reaction was moderate and that the presence of osteoid indicated bone matrix formation.

Apoptosis, proliferation and histomorphometry of spleen were investigated in ovariectomized and non-ovariectomized adult Wistar rats maintained in hypothyroidism induced by daily administration of propylthiouracil (PTU) during 120 days. Two groups ovariectomized euthyroid and non-ovariectomized euthyroid were used as controls. Plasma was collected for free T4 dosage and the spleen for histomorphometry analysis, apoptosis index and the immunohistochemistry expression of caspase 3 and CDC47. Values of free T4 were lower in rats treated with PTU (p

Anatomo-clinical and immunohistochemical findings on three dogs with peripheral nerve sheath tumors assisted at the veterinary hospital are described. Animals showed different clinical signs depending on the location of the tumor. According to microscopy, neoplasia in all the three case were invasive and comprised of pleomorphic, anaplastic with polygonal to very elongated fusiform shapes arranged in a diffuse solid pattern, forming interwoven bundles in some areas. Immunohistochemistry was used to better classify these tumors. The diagnosis of peripheral nerve sheath tumor was confirmed by histological and immunohistochemical findings.

Background Endometriosis is ectopic development of endometrial glands and stroma in extra-uterine sites and if the lesions occur as a well-defined mass is referred to as endometrioma. In the literature, endometrioma has been reported in only women and there are no reports of endometrioma in animals, including non-human primates. Case presentation A rare case of endometrioma is reported in an 11-year-old female German Shepherd with clinical signs of dehydration, anemia and prostration. Necropsy revealed ascites, generalized pallor, and a well-demarcated reddish mass adjacent to the left ovary and uterus and adherent to the retroperitoneum. The mass measured 25.0 × 20.0 cm with intermingled soft and firm areas. Upon incision, the mass was found to be solid with variable sized cystic cavities filled with coagulated blood. Microscopically, the mass was composed of cuboidal or prismatic epithelial cells arranged in tubules or acini. The epithelium of the mass had similar characteristics to the normal endometrium with PAS-positive secretions. The stroma was prominent and formed by loose connective tissue and smooth muscle fibers as confirmed by Masson trichrome. Extensive multifocal areas of hemorrhage were also observed in the stroma of the mass and in the interior of some epithelium-lined, cystic structures. Most of the epithelial cells had strong and diffuse cytokeratin expression, and some had vimentin expression. Epithelial and stromal cells also showed ERβ, AR, VEGF and COX2 expression. The stroma showed areas with strong and diffuse vimentin expression. Factor VIII expression was observed only in the endothelium of blood vessels in the stroma. Conclusions The macroscopic, microscopic and immunohistochemical findings are consistent with an endometrioma.

Morphological and immunohistochemical characterization of angiogenic and apoptotic factors and the expression of thyroid receptors in the ovary of tilapia Oreochromis niloticus in captivity were studied. The morphological evaluation of the ovaries was performed by histological paraffin embedded and stained with HE. The immunohistochemical expressions of CDC47, VEGF, Flk-1, angiopoietin, Tie-2 and thyroid receptor (TRα) were performed by the technique of streptavidein-biotin-peroxidase. Apoptosis was assessed using the TUNEL kit. The relative expression of thyroid hormone receptors (TRα and TRβ) was assessed by RT-PCR real time. The nuclear expression of CDC47 increased with the stage of maturation of the oocyte and was observed in the follicle cells. Apoptotic bodies were observed in the follicular cells of atretic follicles and postovulatory follicles from the ovaries of 150g and 350g fish. Expression of VEGF and its receptor Flk-1 was also observed in the follicular cells, and the expression of both increased with the maturity of the oocyte, with a higher intensity observed in the full-grown follicle. The expression of angiopoietin and of its receptor (Tie 2) was discrete and moderate respectively. TRα expression was independent of follicular development. However, the 350 g tilapia exhibited higher expression of TRβ compared with the 50 g tilapia. We conclude that the proliferative activity and the expression of VEGF and its receptor increase with follicular maturation and that the TRs expression increases with ovarian maturity in tilapia (Oreochromis niloticus). Foram estudadas as caracterizações morfológica e imuno-histoquímica de fatores angiogênicos e apoptóticos e a expressão de receptores tireoidianos no ovário de tilápia Oreochromis niloticus de cativeiro. A avaliação morfológica dos ovários foi realizada por cortes histológicos incluídos em parafina e corados por HE. As expressões imuno-histoquímicas de CDC47, VEGF e seu receptor Flk-1, angiopoetina e seu receptor Tie-2 e recertor tireoidiano (TRα) foram realizadas pela técnica de estreptavideina-biotina-peroxidade. A apoptose foi avaliada utilizando-se kit de TUNEL. A expressão relativa dos receptores de hormônios tireoidianos (TRα e TRβ) foi avaliada pela técnica de RT-PCR tempo real. A expressão nuclear de CDC47 aumentou com a fase de maturação do oócito e foi observada nas células foliculares. Corpos apoptóticos foram observados nas células foliculares de folículos atrésicos e folículos pós-ovulatórios de ovários de peixes com 150g e 350g. A expressão de VEGF e do seu receptor Flk-1 foi também observada nas células foliculares , e a expressão de ambos aumentou com a maturidade do oócito , com uma maior intensidade no folículo maduro. A expressão de angiopoietina e do seu receptor (Tie 2) foi discreta e moderada, respectivamente. A expressão de TRα foi independente do desenvolvimento folicular. No entanto, a tilápia de 350g apresentou maior expressão de TRβ em comparação com a tilápia de 50g. Conclui-se que a atividade proliferativa e a expressão de VEGF e de seu receptor aumenta com a maturação folicular e que a expressão dos TRs aumenta com a maturidade do ovário em tilápia (Oreochromis niloticus).

Background Caffeine is an active alkaloid that can cause damage to bones in formation during prenatal life into adulthood. This compound can pass across the placenta and into the mother’s milk, causing a reduction in bone formation, growth and mass. The objective of this study was to examine the osteogenic potential of osteoblasts extracted from neonatal rats born to mothers treated with caffeine throughout pregnancy. Methods Twenty-four adult Wistar rats were randomly divided into four groups, consisting of one control group and three groups that were treated with 25, 50, or 100 mg/kg of caffeine by an oral-gastric probe throughout the duration of the experimental period (pregnancy). At birth, three puppies from each dam in each group were euthanized, and osteoblasts were extracted from the calvaria of these pups for in vitro testing. Results The osteoblasts extracted from the pups of rats that received 50 mg/kg caffeine during pregnancy exhibited increased expression of osteocalcin, osteopontin, sialoprotein, runx-2, alkaline phosphatase and type I collagen transcripts, resulting in increased synthesis of mineralization nodules. Conclusions Neonates from rats treated with 50 mg/kg caffeine during pregnancy contained osteoblasts with a higher osteogenic potential characterized by increased expression of osteocalcin, osteopontin, sialoprotein, runx-2, alkaline phosphatase and type I collagen and increased synthesis of mineralization nodules.

Foi estudada a relação tireóide-gônadas e sua influência sobre a morfologia óssea de ratas Wistar, com cinco meses de idade, castradas e induzidas ao hipertireoidismo ou mantidas em eutireoidismo por período de 30, 60 e 90 dias. Ratas não castradas foram mantidas nas mesmas condições e serviram como controle. Ao final de cada período, foram determinadas as concentrações plasmáticas de T4 livre, progesterona e estradiol. Os ossos de cada grupo foram submetidos às análises radiológica e histológica. O hipertireoidismo nas ratas não castradas levou à alteração da morfologia do osso, variável ao longo do período experimental, conduzindo, aos 60 dias, à perda de osso trabecular por aumento da reabsorção óssea. Aos 90 dias não houve perda óssea porque o aumento da reabsorção foi acompanhado por maior aposição óssea. Nas ratas eutireóideas castradas, a diminuição dos níveis de progesterona inibiu a aposição óssea, causando, aos 30 dias, pequena perda do osso trabecular das vértebras lombares e do osso alveolar, que se intensificou aos 60 e 90 dias, atingindo também o osso cortical. A administração de tiroxina nas ratas castradas reduziu a osteopenia decorrente da castração aos 60 dias, mas não aos 90 dias, quando a perda óssea foi mais extensa. Conclui-se que o hipoprogesteronismo e o hipoestrogenismo alteram o metabolismo ósseo e que a resposta do osso ao hipertireoidismo depende do perfil plasmático dos esteróides sexuais, do tempo de exposição e da configuração do tecido ósseo.

O efeito do hipotireoidismo sobre o metabolismo ósseo e as paratireóides na deficiência ou suficiência dos esteróides ovarianos foi estudado em 32 ratas Wistar, com 2 meses de idade, distribuídas em 4 grupos de 8: eutireóideo não castrado (ENC), eutireóideo castrado (EC), hipotireóideo não castrado (HNC) e hipotireóideo castrado (HC). Após 120 dias de tratamento, as ratas foram sacrificadas e o plasma colhido para dosagem de T4 livre. Foi evidenciada hipertrofia das paratireóides somente no grupo HNC. As ratas do grupo HNC apresentaram osteopenia de maior extensão e intensidade, decorrente do menor crescimento, da inibição da aposição e do aumento da reabsorção ósseas. Nas ratas EC, a osteopenia foi causada por menor aposição e aumento da reabsorção ósseas. Embora a osteopenia na associação hipotireoidismo-castração tenha sido quase sempre mais intensa em relação à das ratas EC, sua intensidade, quando comparada à osteopenia dos animais HNC, foi variável e dependente do sítio ósseo estudado. Apesar de causar necrose dos ossos de maior metabolismo, a associação hipotireoidismo-castração não potencializou a osteopenia decorrente da ação isolada do hipotireoidismo até os 120 dias de tratamento.

It has been described the anatomopathological and immunohistochemical characteristics of a multinodular neoplasm distributed in the parietal and visceral pleurae, pericardium, thoracic portion of the diaphragm and renal cortex of an eight year-old, female, mixed breed, cat. Based on the anatomopathological and immunohistochemical findings it was firmed the diagnosis of biphasic pleural mesotelioma with renal metastasis.

A relação tireóide-gônadas sobre o perfil hematológico foi investigada em ratas Wistar adultas castradas e não castradas mantidas em hipotireoidismo induzido pela administração diária de propiltiouracil por 120 dias. Dois grupos eutireóideos não castrado e castrado foram mantidos nas mesmas condições e serviram como controle. Foram colhidos o plasma para dosagem de T4 livre e o sangue para análise hematológica. Os valores de T4 livre significativamente menores nas ratas tratadas confirmaram seu estado hipotireóideo. O hipotireoidismo causou anemia apenas em ratas com gônadas funcionais, já que a castração reverteu os efeitos do déficit de tiroxina sobre o eritrograma. Tanto o hipotireoidismo quanto o hipogonadismo apresentaram pouco significado sobre o leucograma.

Apesar da dedicação incessante dos pesquisadores no estudo da osteoporose, muito ainda necessita ser elucidado. A deficiência dos esteróides sexuais, principalmente a de estrógeno, é considerada a principal causa de osteoporose, embora existam inúmeros outros fatores envolvidos. O hipertireoidismo, por exemplo, é considerado um dos fatores de risco para indução ou agravamento da osteoporose e tem despertado o interesse para o estudo dos efeitos de T3 e T4 sobre o metabolismo ósseo. Embora o hipotireoidismo e a afuncionalidade das gônadas seja uma associação freqüente na mulher, a hipofunção da tireóide não é considerada fator de risco para a osteoporose da menopausa. Assim, o estudo da inter-relação entre os distúrbios endócrinos, tão comuns na idade avançada, e a osteoporose é fundamental, pois deste conhecimento poderão advir meios de controle e tratamento adequados, bem como a definição da real natureza do distúrbio ósseo. O objetivo desta revisão é apresentar e discutir alguns aspectos da osteoporose e sua inter-relação com os distúrbios endócrinos da tireóide e das gônadas.

Background Luteal activity is dependent on the interaction of various growth factors, cytokines and hormones, including the thyroid hormones, being that hypo- and hyperthyroidism alter the gestational period and are also a cause of miscarriage and stillbirth. Because of that, we evaluated the proliferation, apoptosis and expression of angiogenic factors and COX-2 in the corpus luteum of hypo- and hyperthyroid pregnant rats. Methods Seventy-two adult female rats were equally distributed into three groups: hypothyroid, hyperthyroid and control. Hypo- and hyperthyroidism were induced by the daily administration of propylthiouracil and L-thyroxine, respectively. The administration began five days before becoming pregnant and the animals were sacrificed at days 10, 14, and 19 of gestation. We performed an immunohistochemical analysis to evaluate the expression of CDC-47, VEGF, Flk-1 (VEGF receptor) and COX-2. Apoptosis was evaluated by the TUNEL assay. We assessed the gene expression of VEGF, Flk-1, caspase 3, COX-2 and PGF2α receptor using real time RT-PCR. The data were analyzed by SNK test. Results Hypothyroidism reduced COX-2 expression on day 10 and 19 (P

The objective of the present study was to evaluate the effect of the thyroid hormones in the gene transcription and immunohistochemical expression of hormonal and angiogenic factors in the placenta of rats. Seventy-two adult female rats were divided equally into propylthiouracil (PTU)-treated, thyroxine (T4)-treated, and control groups. The animals were sacrificed at 10, 14, and 19 days of gestation. We evaluated the immunohistochemical expression of VEGF and its receptor Flk-1. The gene transcription of VEGF, Flk-1, PGF, sFlt1, PL-1, and rPlf was evaluated in placental discs by real-time RT-PCR. The data were analyzed using a Student-Newman-Keuls (SNK) test. At day 10, T4-treated rats presented increased VEGF and PGF gene expression, while PTU-treated rats showed increased rPlf gene expression. Both groups showed reduced Flk-1 and PL-1 gene expression at day 10. At day 14, PTU-treated rats showed reduced VEGF, PGF, and rPlf gene expression. PTU-treated group showed reduced VEGF immunostaining in the placental labyrinth at 14 and 19 days of gestation but it showed increased VEGF immunostaining in the spongiotrophoblast layer at day 14. PTU-treated rats showed increased Flk-1 expression at 14 days of gestation. At days 14 and 19, T4-treated group showed increased PL-1 gene expression and reduced VEGF immunostaining. T4-treated rats also showed reduced Flk-1 and sFlt-1 expression at day 19. Both groups showed increased rPlf gene expression at day 19. In conclusion, rats treated with PTU and T4 have differential effects on the expression of factors involved in placental angiogenic and hormonal activity, and these effects are dependent on the gestational period.

A 10 year old female Boxer was taken dead to veterinary hospital for a necropsy without any clinical history, showing a growth on the dorsal part of the tongue. Based on the histopathological findings mesenchymal chondrosarcoma was diagnosed. This report describes the first case of chondrosarcoma on the base of the tongue in veterinary medicine.

Texto completo: acesso restrito. p.168–170 Submitted by Edileide Reis (leyde-landy@hotmail.com) on 2013-08-27T13:21:01Z No. of bitstreams: 1 1-s2.0-S0737080606000815-main.pdf: 963399 bytes, checksum: 047b7869231c9ae7d603e3b53e7c723c (MD5) Approved for entry into archive by Alda Lima da Silva(sivalda@ufba.br) on 2013-09-03T18:22:13Z (GMT) No. of bitstreams: 1 1-s2.0-S0737080606000815-main.pdf: 963399 bytes, checksum: 047b7869231c9ae7d603e3b53e7c723c (MD5) Made available in DSpace on 2013-09-03T18:22:13Z (GMT). No. of bitstreams: 1 1-s2.0-S0737080606000815-main.pdf: 963399 bytes, checksum: 047b7869231c9ae7d603e3b53e7c723c (MD5) Previous issue date: 2006 A case of renal tubular carcinoma with bone metaplasia is reported in a 20-year-old male, mixed breed horse having a history of weight loss and hematuria. This report includes the clinical signs, macroscopic and microscopic lesions, and the immunohistochemical findings of this neoplasm.

It was reported one case of primary bone lymphoma in eight year-old bitch, Fila, with history of anterior limbs paralysis and side lied. A cervical vertebrae neoplasm was suspected and in view of the poor prognosis, the animal was euthanatized. Based on the anatomopathological and histological findings it was firmed the diagnosis of primary bone lymphoma.

OBJETIVO: Avaliar os efeitos in vitro da cafeína na proliferação, apoptose e expressão de transcriptos gênicos de diferenciação condrogênica na cartilagem de crescimento. MÉTODO: As epífises cartilaginosas de fêmures de ratos neonatos foram divididas em dois subgrupos: os tratados com cafeína e o grupo controle, ambos observados nos tempos de 0, 7, 14 e 21 dias. As epífises cartilaginosas de fêmures de cada subgrupo e de cada tempo foram submetidas à histomorfometria, análise imunoistoquímica, técnica de túnel e RT-PCR em tempo real. RESULTADO: A diminuição da atividade proliferativa e o aumento de condroblastos em apoptose aos 21 dias foram encontrados em ambos os subgrupos. Entretanto a diminuição da proliferação celular causada pela cafeína foi menor quando comparada ao grupo controle e aumentou significativamente a expressão de transcriptos gênicos para diferenciação condrogênica, representada pelo SOX-9 e pelo RUNX-2. Entretanto o cultivo in vitro com cafeína demostrou efeitos antagônicos: apesar dos efeitos positivos na proliferação e diferenciação de condroblatos, cafeína aumentou a apoptose, caracterizada pelo aumento da expressão de caspase-3 e do numero de células em apoptose (p< 0.05). CONCLUSÃO: A cafeína apresenta efeitos antagônicos in vitro na cartilagem em crescimento, aumentando a proliferação, diferenciação e apoptose celular. Estudo experimental. OBJECTIVE: To evaluate the in vitro effects of caffeine on proliferation, apoptosis and gene transcripts expression of chondrogenic differentiation in growth cartilage. METHODS: The cartilaginous epiphyses of femurs of newborn rats, were divided into two subgroups: treated with caffeine and control group, both observed over the time periods of 0, 7, 14 and 21 days. The cartilaginous epiphyses of femurs of each subgroup and each time span were subjected to histomorphometrics, immunohistochemical analysis, tunel technique and RT-PCR in real time. RESULTS: The decrease in proliferative activity and the increase of apoptotic chondroblasts at 21 days were found regardless of the subgroup. However, the decrease in cell proliferation caused by caffeine was lower than in the control group and significantly increased the expression of gene transcripts for chondrogenic differentiation, represented by SOx-9 and RUNx-2. However, the in vitro culture with caffeine revealed antagonistic effects: despite the positive effect on chondroblasts proliferation and differentiation, caffeine increased apoptosis, characterized by increased expression of caspase-3 and of the number of cells undergoing apoptosis (p

The objective of this study was to verify the osteogenic potential of the bone marrow mesenchymal stem cells (MSCs) of ovariectomized and non-ovariectomized female rats with hypo- and hyperthyroidism. Sixty two-month-old female rats were assigned to the following groups: (1) control (sham-operated), (2) ovariectomized (OVX'd), (3) hypothyroid sham-operated (Hypo-), (4) hypothyroid OVX'd, (5) hyperthyroid sham-operated (Hyper-) and (6) hyperthyroid OVX'd. After 135 days of treatment, the female rats were euthanized. We collected plasma to measure the levels of free T4, and the femur for extraction of MSCs. At 7 and 21 days of osteogenic differentiation of MSCs, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) conversion, alkaline phosphatase activity, mineralized nodule number and gene expression for collagen I, osteocalcin, bone sialoprotein and osteopontin were analyzed. The hypothyroid group presented a significant reduction in the osteogenic differentiation of MSCs. The hyperthyroid group did not present changes in the synthesis of mineralized nodules for MSCs at day 21 of differentiation. However, in ovariectomized rats, hyperthyroidism increased the osteogenic differentiation of MSCs characterized by the increase of the alkaline phosphatase activity, the number of mineralized nodules and the expression of osteocalcin, sialoprotein and osteopontin. Our results demonstrated that the hypothyroidism reduces the osteogenic differentiation of MSCs only in non-ovariectomized rats and that the hyperthyroidism increases the osteogenic differentiation of MSCs only in ovariectomized rats.

A rare case of bilateral fusion of a supernumerary kidney was found during the necropsy of a female, 8-year-old, mixed breed cat that died as a result of azotemia and chronic enteritis. Apart from enteritis, necropsy revealed four kidneys, two in the sublumbar left region and two in the sublumbar right region, with cortical and medullary regions well individualized and independent; however, the pelvis was partially fused, giving rise to a single ureter. The kidneys were small, whitish and firm, with irregular surfaces. Microscopically, all kidneys displayed normal renal glomeruli and tubules among the immature renal glomeruli and tubules with characteristics of hypoplasia. Foci of glomerulosclerosis, nephrocalcinosis and interstitial fibrosis were also observed.

Bioactive glass/polymer composites are promising materials for bone tissue engineering. The present research group has developed porous hybrid scaffolds comprised of 50% polyvinyl alcohol/50% bioactive glass with a 70%SiO2–30%CaO composition. Prior studies have also shown the adequate structural and mechanical behavior of these scaffolds. As such, the present study investigates the in vitro and in vivo osteogenic potential of the scaffold, using mesenchymal stem cells (MSC) from the bone marrow of female rats. MTT, alkaline phosphatase activity, collagen secretion and Von Kossa staining were conducted to evaluate the differentiation ability of MSC in an osteogenic medium. The in vitro results indicate an increase in both cell proliferation and osteogenic differentiation when the hybrid material is present. Von Kossa staining showed a progressive increase in mineralization nodules, coupled with time differentiation. For the in vivo evaluation, three groups were studied: (1) group implanted with the hybrid scaffold, (2) group implanted with scaffold colonized by non-differentiated MSC and (3) group implanted with scaffold colonized by differentiated MSC. The scaffolds were subcutaneously implanted on the back of Wistar rats for 1–8 weeks, and histological and histomorphometric analyses were performed. The tissue ingrowth proved to be higher in the groups colonized by MSC in the first week. In the second week, only the hybrid colonized by differentiated MSC presented a larger percentage of connective tissue. In the third, fourth and eighth weeks, all groups presented 70% of the hybrid scaffold filled with tissue. However, only the group with differentiated MSC presented some form of osteoid tissue, indicating that the hybrid scaffold with differentiated MSC does indeed present osteogenic potential. (Some figures may appear in colour only in the online journal)