Kisspeptin-10 Improves Testicular Redox Status but Does Not Alter the Unfolded Protein Response (UPR) That Is Downregulated by Hypothyroidism in a Rat Model.

Hypothyroidism compromises the testicular redox status and is associated with reduced sperm quality and infertility in men. In this regard, studies have demonstrated the antioxidant potential of kisspeptin in reproductive and metabolic diseases. In this study, we evaluate the effects of kisspeptin-10 (Kp10) on the testicular redox, as well as mediators of the unfolded protein response (UPR) in adult rats with hypothyroidism. Adult male Wistar rats were randomly separated into the Control (n = 15), Hypo (n = 13) and Hypo + Kp10 (n = 14) groups, and hypothyroidism was induced with 6-propyl-2-thiouracil (PTU) for three months. In the last month, half of the hypothyroid animals received Kp10. Testis samples were collected for enzymatic, immunohistochemical and/or gene evaluation of mediators of oxidative stress (TBARs, lipid hydroperoxides (LOOH), ROS, peroxynitrite, SOD, CAT and GPX), endoplasmic reticulum stress (GRP78, ATF6, PERK, CHOP, HO-1 and sXBP1) and antiapoptocytes (BCL-2). Hypothyroidism increased apoptosis index, TBARS and LOOH concentrations, and reduced testicular gene expression of Sod1, Sod2 and Gpx1, as well as the expression of Grp78, Atf6, Ho1 and Chop. Treatment with Kp10, in turn, reduced testicular apoptosis and the production of peroxynitrite, while increased SOD1 and GPX ½ expression, and enzymatic activity of CAT, but did not affect the lower expression of UPR mediators caused by hypothyroidism. This study demonstrated that hypothyroidism causes oxidative stress and dysregulated the UPR pathway in rat testes and that, although Kp10 does not influence the low expression of UPR mediators, it improves the testicular redox status, configuring it as an important antioxidant factor in situations of thyroid dysfunction.

Kisspeptin (Kp-10) inhibits in vitro osteogenic differentiation of multipotent mesenchymal stromal cells extracted from the bone marrow of adult rats.

Kisspeptin (Kp-10) is a neuropeptide that binds to GPR54 receptors, exerting several functions mainly in the nervous and reproductive systems of the body. However, its effects and mechanisms of action on the skeletal system remain poorly understood. This study evaluated the effects of different concentrations of Kp-10 on in vitro osteogenic differentiation of multipotent mesenchymal stromal cells (MSCs) extracted from the bone marrow (BM) of adult Wistar rats. Two-month-old female rats were euthanized to extract BM from long bones to obtain MSCs. Four experimental groups were established in vitro: a control and Kp-10 at concentrations of 0.01, 0.05 and, 0.1 µg/mL. After induction of osteogenic differentiation, cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl tetrazolium bromide (MTT) assay, alkaline phosphatase activity, collagen synthesis, percentage of area covered by MSCs/field and mineralized nodules/field, and immunocytochemistry of the GPR54 receptor tests. Furthermore, evaluation of gene transcripts for type I collagen, Runx-2, Bmp-2, bone sialoprotein, osteocalcin and osteopontin was performed using real-time RT-qPCR. It was observed that MSCs expressed GPR54 receptor to which Kp-10 binds during osteogenic differentiation, promoting a negative effect on osteogenic differentiation. This effect was observed at all the Kp-10 concentrations in a concentration-dependent manner, characterized by a decrease in the activity of alkaline phosphatase, collagen synthesis, mineralized nodules, and decreased expression of gene transcripts for type I collagen, osteocalcin, osteopontin, and Runx-2. Thus, Kp-10 inhibits in vitro osteogenic differentiation of MSCs extracted from the BM of adult Wistar rats.

Tracheal stenosis in a yellow-crowned parrot (Amazona ochrocephala) due to diffuse ossification and osteopetrosis of tracheal rings.

Tracheal luminal stenosis can cause clinical respiratory distress in wild birds. We describe a case of tracheal stenosis due to diffuse ossification with osteopetrosis of tracheal rings in a yellow-crowned parrot (Amazona ochrocephala) with a history of chronic respiratory distress and death after development of marked dyspnoea. An ante-mortem radiographic examination revealed that the tracheal rings were radiopaque and that there were multiple areas of osteopenic change in long bones. At necropsy, there was stenosis of the tracheal rings characterized by complete replacement of cartilage by thickened compact bone with osteopetrosis and bone necrosis. The clinical respiratory distress and death of the parrot were associated with tracheal luminal stenosis due to thickening of the tracheal rings by diffuse ossification with osteopetrosis.

Effect of the addition of different concentrations of Solanum glaucophyllum desf. extract on chondrocyte cultures from the growth cartilage of newborn rats.

Solanum glaucophyllum Desf. is a calcinogenic plant responsible for enzootic calcinosis that affects ruminants and causes alterations in bone and cartilaginous tissues, among others. It is believed that changes in cartilage tissue, with reduced bone growth, are due to hypercalcitoninism, caused by excess vitamin D. However, we hypothesized that S. glaucophyllum Desf. can act directly on chondrocytes and therefore, chondrocyte cultures from the epiphysis of the long bones of newborn rats were used as a model to elucidate the direct effects of S. glaucophyllum Desf. on bone growth. Plant samples were collected from Cañuelas, Argentina. An aliquot of the plant extract was used to quantify vitamin D (1,25(OH)2D3). The effects of the three concentrations of the plant extract were tested in cultures of chondrocytes extracted from the epiphyses of the long bones of 32 three-day-old Wistar rats. A control group (without extract), and three groups treated with different concentrations of plant extract were formed: group 1 (100 µL/L); group 2 (1 mL/L), and group 3 (5 mL/L), containing respectively 1 × 10-9 M, 1 × 10-8 M, and 5 × 10-8 M of 1,25(OH)2D3. After 7, 14, and 21 days of culture, MTT assay for cell viability, alkaline phosphatase activity, and quantification of the percentage of areas with glycosaminoglycans (GAG) stained with periodic acid-Schiff (PAS) were performed. On day 7, all chondrocytes in group 3, that is, those with the highest concentration of plant extract, died. On days 14 and 21, groups 1 and 2 showed a significant reduction in chondrocyte viability compared to the control. At 7, 14, and 21 days, groups 1 and 2 showed significantly lower alkaline phosphatase activity than the control. On day 21, group 2 showed a significant reduction in areas with PAS + GAGs. There were no significant differences between the groups in the expression of gene transcripts for Sox9, Col2, ColX, and aggrecan. The S. glaucophyllum Desf. extract directly affected growing rat chondrocytes by reducing viability, alkaline phosphatase activity, and GAG synthesis without altering the expression of gene transcripts for Sox9, Col2, ColX, and aggrecan, which may be one of the mechanisms by which there is a reduction in bone growth in animals intoxicated by the plant.

Maternal hyperthyroidism alters the immunological mediators profile and population of natural killers cells in decidua of rats.

Decidual immunological mediators modulate placental formation, decidualization and fetal development. However, the effect of maternal hyperthyroidism on decidual immunology needs further research. The aim of this study was to evaluate the population of uterine natural killer cells (uNKs) and the expression of immunological mediators in the decidua of female rats throughout pregnancy. Wistar rats were used and hyperthyroidism was induced by daily administration of L-thyroxine (T4) throughout pregnancy. The population of uNK cells in decidua was evaluated by immunostaining Lectin DBA, as well as the expression of interferon γ (INFγ), macrophage migration inhibitory factor (MIF), interleukin 15 (IL-15) and inducible nitric oxide synthase (iNOS) at 7, 10, 12, 14 and 19 days of gestation (DG). Maternal hyperthyroidism reduced the DBA+ uNK cell population in the decidua at 7 (P < 0.05) and 10 (P < 0.01) DGs compared to that in the control group, while it increased in the basal decidua (P < 0.05) and metrial gland (P < 0.0001) at the 12th DG. Hyperthyroidism also increased immunostaining of IL-15 (P < 0.0001), INFγ (P < 0.05), and MIF (P < 0.05) in the 7th DG, and increased immunostaining of IL-15 (P < 0.0001) and MIF (P < 0.01) in the 10th DG. However, excess thyroxine reduced IL-15 expression in the metrial gland and/or basal decidua in the 12th (P < 0.05), 14th (P < 0.01), and 19th (P < 0.001) DGs, as was also observed for INFγ in the basal decidua (P<0.001) and metrial gland (P < 0.0001) in the 12th DG. Regarding iNOS, an antiinflammatory cytokine, lower expression was observed in the basal decidua of hyperthyroid animals at 7 and 12 DGs (P < 0.05), whereas an increase occurred in the 10th DG (P < 0.05). These data demonstrate that maternal hyperthyroidism in female rats, particularly between 7 and 10 DGs, reduces the population of DBA+ uNKs in the decidua and increases the expression of inflammatory cytokines, suggesting a more proinflammatory environment in early pregnancy caused by this gestational disease.

Microscopic Alterations of the Dental Pulp in Surgically Extracted Teeth of Dogs.

Microscopic alterations in the dental pulp of dogs have not been extensively studied. The aim of this study was to investigate microscopic alterations of the dental pulp in dogs' teeth. One hundred and ten surgically extracted teeth (20 incisors, 23 canines, 28 premolars, and 39 molars) from 74 dogs, of different ages, with a history of chronic periodontitis (66 dogs), periapical abscesses (2 dogs), pulpitis (2 dogs), oral cavity neoplasms (2 dogs), dens invaginatus (1 dog), and dental fractures (1 dog) were included. Eight-one maxillary and 29 mandibular teeth were included. Coronal, radicular, and coronal plus radicular calculus were present in 28.2%, 17.3%, and 54.5% of the teeth, respectively. In total 78 teeth (71%) had pulp alterations, including fibrosis (26%), calcification (14%), necrosis associated with the absence of odontoblasts (14%), presence of predentin and dentin inside the cavity (8%), odontoblastic hyperplasia (3%), pigmentation (3%), pulpitis (2%), and pulp stones (1%). Forty-nine (60.5%) of the maxillary teeth and all of the mandibular teeth had pulp alterations. The premolars were most affected, and the molars least affected, by pulp alterations. Pulp fibrosis, calcification, and necrosis were observed in teeth irrespective of the distribution of dental calculus.

Three Cases of Mandibular Ossifying Fibroma in Cattle.

Neoplastic masses were evaluated in the rostral region of the mandible of three young adult cattle. In all three cases, the masses were macroscopically large, firm, ulcerated, infiltrative, whitish and solid, and led to tooth displacement and loss. Radiographically, the neoplastic masses were intraosseous and radiolucent with foci of radiopacity. Loss of radiopacity due to bone necrosis was seen in the mandibular bone adjacent to the neoplasms. Histologically, well-differentiated, infiltrative non-encapsulated mesenchymal neoplastic proliferation replaced the mandibular bone and extended to the oral mucosa in all three cases. The neoplastic cells had scant cytoplasm and fusiform or oval hyperchromatic nuclei with loose chromatin, and were arranged in bundles in various directions. Within the neoplastic tissue, there were mineralized bone trabeculae and unmineralized osteoid, lined by a layer of osteoblasts and osteocytes within the lacunae. The pre-existing bone tissue adjacent to the neoplasm had areas of necrosis and osteoclasis of variable extent and intensity. Based on the macroscopic, radiographic and microscopic findings, a diagnosis of mandibular ossifying fibroma was established in all three cattle.

Kisspeptin treatment improves fetal-placental development and blocks placental oxidative damage caused by maternal hypothyroidism in an experimental rat model.

Maternal hypothyroidism is associated with fetal growth restriction, placental dysfunction, and reduced kisspeptin/Kiss1R at the maternal-fetal interface. Kisspeptin affects trophoblastic migration and has antioxidant and immunomodulatory activities. This study aimed to evaluate the therapeutic potential of kisspeptin in the fetal-placental dysfunction of hypothyroid Wistar rats. Hypothyroidism was induced by daily administration of propylthiouracil. Kisspeptin-10 (Kp-10) treatment was performed every other day or daily beginning on day 8 of gestation. Feto-placental development, placental histomorphometry, and expression levels of growth factors (VEGF, PLGF, IGF1, IGF2, and GLUT1), hormonal (Dio2) and inflammatory mediators (TNFα, IL10, and IL6), markers of hypoxia (HIF1α) and oxidative damage (8-OHdG), antioxidant enzymes (SOD1, Cat, and GPx1), and endoplasmic reticulum stress mediators (ATF4, GRP78, and CHOP) were evaluated on day 18 of gestation. Daily treatment with Kp-10 increased free T3 and T4 levels and improved fetal weight. Both treatments reestablished the glycogen cell population in the junctional zone. Daily treatment with Kp-10 increased the gene expression levels of Plgf, Igf1, and Glut1 in the placenta of hypothyroid animals, in addition to blocking the increase in 8-OHdG and increasing protein and/or mRNA expression levels of SOD1, Cat, and GPx1. Daily treatment with Kp-10 did not alter the higher protein expression levels of VEGF, HIF1α, IL10, GRP78, and CHOP caused by hypothyroidism in the junctional zone compared to control, nor the lower expression of Dio2 caused by hypothyroidism. However, in the labyrinth zone, this treatment restored the expression of VEGF and IL10 and reduced the GRP78 and CHOP immunostaining. These findings demonstrate that daily treatment with Kp-10 improves fetal development and placental morphology in hypothyroid rats, blocks placental oxidative damage, and increases the expression of growth factors and antioxidant enzymes in the placenta.

Maternal Hyperthyroidism in Rats Alters the Composition and Gene Expression of the Matrix Produced In Vitro by Chondrocytes from Offspring with Intrauterine Growth Restriction.

Herein, we aimed to evaluate cultures of femoral chondrocytes from offspring of rats with intrauterine growth restriction (IUGR) induced by maternal hyperthyroidism. Fourteen adult female Wistar rats were divided into two groups, a control group and a group treated with daily L-thyroxine administration using an orogastric tube (50 µg/animal/day) during pregnancy. Three days after birth, the offspring were euthanized for chondrocyte extraction. At 7, 14, and 21 days, viability and alkaline-phosphatase (ALP) activity were assessed using the MTT assay and BCIP/NBT method, respectively, in a 2D culture. Pellets (3D cultures) were stained with periodic acid Schiff (PAS) to assess the morphology and percentage of PAS+ areas. The gene transcripts for Col2, Col10, Acan, Sox9, and Runx2 were evaluated by qRT-PCR. The MTT and ALP-assay results showed no significant differences between the groups. Maternal hyperthyroidism did not alter the chondrocyte morphology, but significantly reduced the percentage of PAS+ areas, decreased the expression of the gene transcripts of Col2 and Acan, and increased Sox9 expression. Maternal hyperthyroidism in rats alters the composition and gene expression of the matrix produced by chondrocytes from offspring with IUGR. This may be one of the mechanisms through which excess maternal thyroid hormones reduce offspring bone growth.

Maternal hypothyroidism reduces the expression of the kisspeptin/Kiss1r system in the maternal-fetal interface of rats.

Alterations of circulating and placental levels of kisspeptin have been associated with gestational diseases. However, there are still no studies on the placental and decidual expression of Kiss1 and its receptor Kiss1r in maternal hypothyroidism, which is the aim of this work. We demonstrate that the fetoplacental restriction caused by hypothyroidism in rats is associated with a reduction in the Kiss1r expression and reduced Kiss1 and Kiss1r mRNA levels in the decidua and/or placenta. This demonstrate that fetoplacental restriction in hypothyroid rats is linked with a suppression of the kisspeptin/Kiss1r system at the maternal-fetal interface.

Phenotype and synthesis activity of joint chondrocytes extracted from newborn rats with prenatal ethanol exposure.

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.

Effect of ethanol consumption during pregnancy and lactation on bone histomorphometry and in vitro osteogenic differentiation of bone marrow mesenchymal stem cells in maternal rats.

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.

Dentinal Lesions in Dogs with Dental Calculus.

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.

Kisspeptin/Kiss1r system and angiogenic and immunological mediators at the maternal-fetal interface of domestic cats.

The Kisspeptin/Kiss1r system is a key regulator of reproduction by stimulating gonadotrophin-releasing 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.

Ethanol Alters Phenotype and Synthesis Activity of Rat Neonatal Articular Chondrocytes Grown in 2- and 3-Dimensional Culture.

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.

Pyometra and estrous cycle modulate the uterine expression of the kisspeptin system and angiogenic and immune factors in cats.

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.

Differentiation of Mesenchymal Stem Cells from Humans and Animals into Insulin-producing Cells: An Overview In Vitro Induction Forms.

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.

Use of bone marrow for detection of toxic chemicals for the elucidation of poisoning in forensic veterinary medicine / Uso de medula óssea para detecção de produtos químicos tóxicos para a elucidação de intoxicação em patologia veterinária forense

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.(AU)

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.(AU)

In vitro effects of the co-release of icariin and strontium from bioactive glass submicron spheres on the reduced osteogenic potential of rat osteoporotic bone marrow mesenchymal stem cells.

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.

The Solanum glaucophyllum Desf. extract reduces mineralized matrix synthesis in osteogenically differentiated rat mesenchymal stem cells in vitro.

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.