Avaliação da eficácia do sistema rigeneracon no tratamento de lesões de calvária em ratos / [Evaluation of the efficacy of the rigeneracon system in the treatment of calvary injuries in rats]

Defeitos ósseos constituem um problema de saúde global. O sistema Rigenera permite a extração de microenxertos ricos em células-tronco mesenquimais (CTMs). Objetivou-se avaliar o processo de regeneração óssea por enxertos obtidos pelo sistema Rigenera em defeitos críticos na calvária de ratos. Foram utilizados 18 ratos Wistar, machos, pesando 285±29g, distribuídos em três grupos (n=6), sendo cada animal controle de si mesmo, denominados G15-Controle e G15-Tratado (15 dias); G30-Controle e G30-Tratado (30 dias) e G60-Controle e G60-Tratado (60 dias). Foram realizadas duas lesões de 5mm de diâmetro em cada antímero da calvária. Nos grupos tratados, foram utilizados microenxertos autólogos de cartilagem xifoide, obtidos pelo sistema Rigenera. O defeito contralateral serviu como controle em todos os animais. Os animais foram eutanasiados aos 15, 30 e 60 dias após a cirurgia, e as amostras foram processadas para a histoquímica. Nos grupos controle, não foram observados sinais de regeneração óssea, enquanto nos grupos tratamento foram verificadas áreas de formação óssea e tecido mesenquimal ativado. O sistema Rigenera foi eficiente na obtenção de microenxertos autólogos, para terapia celular em defeito crítico de calvária de ratos. Com o aprimoramento do protocolo, o sistema Rigenera poderá ser amplamente utilizado no tratamento de lesões ósseas.(AU)

Bone defects are a global health problem. The Rigenera system allows the extraction of micro grafts rich in mesenchymal stem cells (MSCs). The objective of this study was to evaluate the bone regeneration process by grafts obtained by the Rigenera system in defects in the rats calvarian. Eighteen male Wistar rats were used, weighing 285 ± 29g, distributed in three groups (n = 6), where each animal was treatment and control, called G15-Control and G15-Treated (15 days); G30-Control and G30-Treated (30 days) and G60-Control and G60-Treated (60 days). Two 5mm diameter lesions were performed on each calvaria side. In the treated groups, autologous micrograft from xiphoid cartilage, obtained by the Rigenera system, were used. The other defect served as a control in all animals. The animals were euthanized at 15, 30 and 60 days after the surgery and the samples were processed for histochemistry. In the control groups, no signs of bone regeneration were observed, while in the treatment groups, areas of bone formation and activated mesenchymal tissue were verified. The Rigenera system was efficient in obtaining autologous micrograft for cell therapy in a critical calvaria defect in rats. Rigenera system can be widely used in the treatment of bone injuries.(AU)

Ação do estanozolol sobre a histologia renal e hepática em ratos treinados com natação / [Stanozolol action on renal and hepatic histology in swimming trained rats]

No presente estudo, foram analisados os efeitos do estanozolol, associado ou não à atividade física, sobre o hemograma, o peso ponderal, a ingestão líquida e sólida, a urinálise, a expressão do VEGF-A renal e o glicogênio hepático, além da histopatologia hepática e renal em ratos Wistar. Foram utilizados 32 ratos Wistar, machos, jovens, separados em quatro grupos: GC (grupo controle); GCE (grupo controle-exercício); GT (grupo tratamento-esteroide); GTE (grupo tratamento-esteroide-exercício). Os animais dos grupos GT e GTE (n=16) foram submetidos a injeções subcutâneas, cinco dias/semana, durante 30 dias, na concentração de 5mg/kg de estanozolol diluído em 1mL de óleo de gergelim, utilizado como veículo. A natação foi definida como exercício físico. Houve aumento no peso dos animais submetidos ao estanozolol e ao exercício a partir da terceira semana de uso e aumento da excreção urinária a partir da quinta semana; os demais parâmetros da urinálise foram semelhantes entre os grupos. O uso de estanozolol associado ou não à atividade física promoveu redução da expressão do VEGF-A nos rins e do glicogênio hepático, além de alterações histopatológicas nesses órgãos. Quanto à hematologia, houve uma diminuição dos leucócitos no GTE em relação aos grupos GT e GCE. Quanto aos linfócitos, houve um aumento no GT e uma diminuição no GTE, e, em relação ao número de plaquetas, houve diminuição no GTE quando comparado ao GT e ao GCE Assim, conclui-se que estanozolol na dose de 5,0mg/kg causa alterações renais e hepáticas em ratos Wistar, podendo levar à falência dos rins e do fígado.(AU)

The goal of this study was to determine the effect of stanozolol (ST) on kidney and liver of Wistar rats. Thirty-two male animals were divided into the following four groups: control group (CG); Control group-exercise (GCE); Group-steroid treatment (GT); Group treatment-steroid-exercise (GTE). Swimming was defined as exercise. The animals GT and GTE was submitted to subcutaneous injections, five days/week for 30 days, at a concentration of 5mg/kg ST diluted in 1mL/kg of sesame oil. The results showed an increase in weight gain in all animals submitted to ST and exercise from the 3rd week of use and increase in urinary excretion from the 5th week and the other urinalysis parameters were similar. The ST associated or not with physical activity reduced VEGF-A expression in the kidneys and hepatic glycogen, as well as histopathological changes in these organs. Regarding hematology, there was a decrease in leukocytes in the GTE. As for lymphocytes there was an increase in GT and a decrease in GTE, and in relation to the number of platelets, there was a decrease in GTE. In conclusion, the administration of stanozolol at 5.0mg/kg caused a structural change of kidney and liver in treated animals.(AU)

Influência da somatotropina recombinante bovina no desenvolvimento folicular e na coleta de embriões em éguas / Influence of bovine recombinant somatotropin in the follicular development and embryo colletion in mares

Dez éguas, sem raça definida, foram submetidas a avaliações ultrassonográficas durante o intervalo interovulatório, avaliando-se folículos ≥ 5mm. Cinco éguas foram tratadas com 500mg de r-bST no primeiro e no 14º dia pós-ovulação (grupo GT), e as demais com soro fisiológico (grupo GC). Quando o folículo dominante atingiu diâmetro ≥ 40mm, foram induzidas com hCG e inseminadas 24 horas após, sendo submetidas à coleta de embrião seis dias após a ovulação. Os dados foram agrupados de acordo com o diâmetro do folículo dominante nas fases de emergência, divergência, dominância, pré-ovulatória, indução, inseminação e ovulação. Todas as éguas foram usadas duas vezes, no mesmo grupo. O GT apresentou crescimento folicular precoce para as fases de emergência, divergência, dominância e pré-ovulatória, assim como para o seu maior folículo subordinado, que cresceu mais cedo. As taxas de recuperação foram de 90% (GC) e 70% (GT), em 16 estruturas coletadas, obtendo-se uma não fecundada e um blastocisto inicial para o grupo GC; os demais, no estágio de mórula, apresentaram comportamento semelhante entre os grupos. Conclui-se que a r-bST influencia a dinâmica folicular de éguas, levando a uma antecipação do desenvolvimento folicular, que pode ser utilizada para encurtar o ciclo estral.(AU)

Ten undefined mare breeds were submitted to ultrasonographic evaluations during the interovulatory interval, evaluating follicles measuring ≥ 5mm. Five mares were treated with 500mg r-bST on the first and the 14th day after ovulation (TG group), and the others with saline (CG group). When the dominant follicle reached a diameter ≥ 40mm the ovulation was induced with hCG, and the mares were inseminated 24 hours later and submitted to embryo collection six days after ovulation. The data were grouped according to the diameter of the dominant follicle in the emergence, divergence, dominance, preovulatory, induction, insemination and ovulation phases. All mares were used twice, in the same group. The GT showed early follicular growth for the emergence, divergence, dominance and pre-ovulatory phases, as well as for its greater subordinate follicle, growing earlier. The recovery rates were 90% (CG) and 70% (TG), and 16 structures were collected, obtaining an unfertilized embryo and an initial blastocyst for the CG group, the others in the morula stage behaved similarly between the groups. It can be concluded that r-bST influences the follicular dynamics of the mares, leading to an anticipation of the follicular development that can be used to shorten the estrous cycle.(AU)

Efeito da dexametasona e melatonina exógenas sobre parâmetros sanguíneos, progesterona, carboidratos totais e histomorfometria de órgãos em ratas prenhes / The effect of dexamethasone and exogenous melatonin on blood parameters, progesterone, total carbo hydrates and histomorphometry of organs in pregnant rats

A dexametasona é utilizada em casos de gestação com risco de prematuridade; porém, doses suprafisiológicas podem afetar a embriogênese. A melatonina tem demonstrado prevenir efeitos deletérios dos glicocorticoides. Assim, avaliamos a influência da melatonina sobre efeitos da dexametasona em ratas prenhes através dos seguintes parâmetros: 1. Hemograma e perfil glicídico; 2. Níveis de progesterona; e 3. Histomorfometria e histoquímica. Foram utilizadas 20 ratas divididas nos grupos: I - ratas prenhes que receberam placebo (Controle); II - ratas prenhes tratadas com dexametasona (0,8mg/kg); III - ratas prenhes tratadas com melatonina (0,5mg/kg); IV - ratas prenhes tratadas com dexametasona e melatonina. Todos os tratamentos foram iniciados 10 dias após confirmação do acasalamento até o final da prenhez. O sangue foi coletado no 7º, 14º e 21º dia de prenhez. As dosagens de carboidratos e progesterona foram realizadas pelo método antrona e ELISA, respectivamente. O fígado, rins e adrenais foram analisados histoquímica e morfometricamente. No 7º dia de prenhez, não houve alteração significativa nos parâmetros analisados. Porém, no 14º dia de prenhez, houve aumento significativo do volume de hematócrito, redução do número total de hemácias e leucócitos, neutrofilia, linfopenia, eosinopenia e redução do diâmetro das hemácias nas matrizes tratadas com dexametasona. Esses efeitos permaneceram no 21º dia de prenhez, exceto para o hematócrito, o qual reduziu. Verificou-se ainda redução significativa dos níveis de glicose (21º dia de prenhez) e progesterona (14º e 21º dia de prenhez). Não houve alteração nos parâmetros morfométricos e histoquímico no fígado, rins e adrenais. A dexametasona na dosagem de 0,8mg/kg, administrada a partir do terço médio da prenhez, produz alterações hematológicas, bioquímicas e hormonais em ratas, sendo prevenidas pela melatonina; porém não afeta o fígado, rins e adrenais quanto aos parâmetros morfométricos e histoquímicos.

Dexamethasone is used in cases of pregnancy with prematurity risk. However, it may affect the embryogenesis when used in supraphysiological doses. Melatonin has been shown to prevent the deleterious effects of glucocorticoids. Therefore, the influence of melatonin on the effects of dexamethasone on pregnant rats was evaluated through the following parameters: 1. Hemogram and glucose profile; 2. Progesterone levels; 3. Histomorphometry and histochemistry analyses. Twenty female rats were divided into the following groups: I - rats that received placebo (Control); II - rats treated with dexamethasone (0.8mg/kg); III - rats treated with melatonin (0.5mg/kg); IV - rats treated with dexamethasone and melatonin. All treatments started 10 days after confirmation of mating and lasted until the end of the pregnancy. Blood samples were collected on the 7th, 14th, and 21st day. Carbohydrate and progesterone levels were determined with the antrona and ELISA method, respectively. The liver, kidneys, and adrenal glands were analyzed morphometrically and histochemically. On the 7th day of pregnancy there were no significant changes in the parameters analyzed. However, at 14 days of pregnancy there was a significant increase of hematocrit, reducing the total number of erythrocytes and leukocytes, neutrophilia, lymphopenia, eosinopenia and reduced diameter of red blood cells in rats treated with dexamethasone. These effects remained on the 21st of day of pregnancy, except for the hematocrit, which was reduced. There was also a significant reduction in glucose levels (21st day) and progesterone (14th and 21st days). There was no change in the histochemical and morphometric parameters in the liver, kidneys and adrenals. Dexamethasone at a dosage of 0.8mg/kg administered from the middle third of pregnancy produces hematological, biochemical and hormonal changes in rats, being prevented by melatonin, but does not affect the liver...

Ação da melatonina sobre a dinâmica sanguínea de ratas prenhes e sobre a histogênese do baço e do timo da prole / The action of melatonin on the blood dynamics of pregnant rats and on the histogenesis of the spleen and thymus of offspring

Investigou-se a influência da melatonina sobre o hemograma de ratas prenhes e dos filhotes e sobre a histogênese e morfometria do baço e do timo dos filhotes. A melatonina foi administrada na dose 0,5mg/kg de peso corporal, dissolvida em 0,1mL de etanol e diluída em 0,3mL de solução salina. Para análise do hematócrito, contagem total de hemácias e contagem total e diferencial dos leucócitos, amostras de sangue foram coletadas no sétimo, 14ºe 21ºdias de prenhez e aos 10 dias de nascimento dos filhotes. Cortes histológicos do baço e do timo da prole foram utilizados para histoquímica e morfometria. A ausência da melatonina promoveu alterações no hemograma apenas no terço final da gestação, sem interferir no hemograma dos filhotes, e induziu modificações morfológicas e morfométricas no timo e no baço nos primeiros dias de vida dos filhotes. Concluiu-se que a melatonina materna é importante para a modulação do hemograma em ratas prenhes e para o desenvolvimento normal do baço e do timo dos filhotes.

We investigated the influence of melatonin on the hemogram of pregnant rats and puppies, and on the histogenesis and morphology of the spleen and thymus of puppies. Melatonin was administered at a dose 0.5mg/kgbody weight, dissolved in 0.1mL of ethanol and diluted in 0.3mL of saline. For hematocrit analysis, total erythrocyte count and total and differential leukocyte count, blood samples were collected on the 7th, 14th and 21st days of pregnancy and the offspring at 10 days of birth. Histological sections of spleen and thymus of the offspring were used for histochemistry and morphometry. The absence of melatonin promoted changes in blood count only in the final third of gestation, without interfering with the hemogram of the puppies, and induced morphological and morphometric changes in the thymus and spleen in the first days of life of the puppies. It was concluded that maternal melatonin is important for the modulation of the blood count in pregnant rats and the normal development of the spleen and thymus of the offspring.

Efeito da administração pré-natal da dexametasona em ratas sobre os perfis glicídicos e hematológicos materno e da prole / Effect of prenatal administration of dexamethasone in rats on profiles hematologic and glicidic maternal and offspring

Avaliou-se o efeito da administração de dexametasona no início e no meio da gestação de ratas, sobre os perfis glicídicos e hematológicos materno e da prole. Os animais foram submetidos aos seguintes tratamentos: dexametasona do primeiro ao sétimo dia e placebo do oitavo ao 14º dia; placebo do primeiro ao sétimo dia e dexametasona do oitavo ao 14º dia; dexametasona do primeiro ao 14º dia e placebo do primeiro ao 14º dia de gestação. A dexametasona foi administrada por via intraperitoneal, na dose de 0,8mg/kg. Foram coletadas amostras de sangue no sétimo, 14º e 21º dias de gestação, e de sangue e tecido hepático da prole no quinto, 10º e 15º dias pós-natal. Para a verificação das reservas de glicogênio hepático da prole, cortes histológicos foram corados pelo ácido periódico de Schiff. Os resultados apontam para um efeito tempo-dependente da administração de dexametasona durante a gestação, levando a alterações temporais distintas na hematologia e na concentração plasmática de carboidratos nas matrizes e na prole.

The effect of the administration of dexamethasone at the beginning and middle of the pregnancy in rats on hematological and glicidic maternal and offspring profile was evaluated. The animals underwent the following treatments: dexamethasone from the first to the seventh day and placebo from the 8th to the 14th day, placebo from the first to the seventh day and dexamethasone from the 8th to the 14th day; dexamethasone from the 1st to the 14th day, and placebo treatment from the first to the 14th day of gestation. Dexamethasone was administered intraperitoneally at a dose of 0.8 mg/kg. Blood samples were collected on the 7th, 14th and 21st days of pregnancy, and blood and liver tissue of offspring on the fifth, 10th and 15th days postnatal. For verification of the reserves of liver glycogen on the offspring, histological sections were stained with periodic acid-Schiff. The results point to a time-dependent effect of dexamethasone during pregnancy, leading to different temporal changes in hematology and plasma levels of carbohydrates in headquarters and in the offspring.

The lipid peroxidation end-product 4-HNE induces COX-2 expression through p38MAPK activation in 3T3-L1 adipose cell.

Oxidative stress and low grade chronic inflammation are increased in accumulating fat. Our objective was to test whether 4-hydroxynonenal (4-HNE), an end-product of lipid peroxidation, affects cyclooxygenases in 3T3-L1 adipose cells. 4-HNE increased COX-2 mRNA and protein expression and p38MAP-kinase phosphorylation in a dose-dependent manner. Pretreatment of 3T3-L1 cells by a selective inhibitor of p38MAPK (PD 169316) abolished 4-HNE and glucose oxidase induced COX-2 expression. Our results show that oxidative stress induces COX-2 expression through the production of 4-HNE which activates p38MAPKinase, suggesting that 4-HNE links oxidative stress and chronic inflammation through the activation of cyclooxygenase.

Covalent binding of 15-deoxy-delta12,14-prostaglandin J2 to PPARgamma.

Since 15-deoxy-delta(12,14)-prostaglandin J(2) (15dPGJ(2)) has been identified as an endogenous ligand of PPARgamma thus inducing adipogenesis, it has been reported to play active parts in numerous cellular regulatory mechanisms. As 15dPGJ(2) has been shown to covalently bind several peptides and proteins, we investigated whether it also covalently binds PPARgamma. We first observed that after incubation of 15dPGJ(2) with recombinant PPARgamma, the quantity of free 15dPGJ(2) measured was always lower than the initial amount. We then measured the ability of the labeled agonist rosiglitazone to displace the complex PPARgamma(2)/15dPGJ(2) obtained after pre-incubation. We observed that the binding of rosiglitazone was dependent on the initial concentration of 15dPGJ(2). Finally using MALDI-TOF mass spectrometry analysis, after trypsinolysis of an incubate of the PPARgamma(2) ligand binding domain (GST-LBD) with 15dPGJ2, we found a fragment (m/z = 1314.699) corresponding to the addition of 15dPGJ(2) (m/z = 316.203) to the GST-LBD peptide (m/z = 998.481). All these observations demonstrate the existence of a covalent binding of 15dPGJ(2) to PPARgamma, which opens up new perspectives to study the molecular basis for selective activities of PPARs.

Effects of oxidative stress on adiponectin secretion and lactate production in 3T3-L1 adipocytes.

Obesity is an increasing nutritional disorder in developed countries, and oxidative stress has been identified as a key factor in numerous pathologies such as diabetes, inflammation, and atherosclerosis, which are favored by obesity. The objective of the present study was to investigate the effects of oxidative stress in 3T3-L1 adipose cells on two parameters involved in metabolic complications associated with obesity, namely adiponectin secretion and lactate production. Differentiated 3T3-L1 adipose cells were exposed to increasing concentrations of glucose oxidase. 4-Hydroxynonenal (4-HNE), a relevant lipid peroxidation by-product which may affect several metabolic processes in making covalent adducts with various molecules; adiponectin secretion; and lactate production were measured in response to glucose oxidase exposure. Results show an inhibition of adiponectin mRNA expression by glucose oxidase and a significant inverse correlation between 4-HNE formation and adiponectin secretion. Furthermore, 4-HNE alone inhibits adiponectin production by 3T3-L1. On the other hand, glucose oxidase and 4-HNE significantly stimulated lactate production by 3T3-L1 adipocytes. These results demonstrate that adipose cells are highly sensitive to oxidative stress, with subsequent decreased adiponectin secretion and increased lactate production, two events involved in the development of insulin resistance.

Biochemical and morphological changes in the liver after hepatic artery ligation in the presence or absence of extrahepatic cholestasis.

The present study was carried out to investigate the biochemical and morphological changes in the liver after ligation of the hepatic artery (HA) in the presence and in the absence of extrahepatic cholestasis (EHC). The study was conducted on 100 rats divided into four groups of 25 animals each: group 1, sham operation; group 2, hepatic artery ligation (HAL); group 3, bile duct ligation (BDL); and group 4, HAL plus BDL. All animals were sacrificed 7 days after surgery when total bilirubin and fractions, alkaline phosphatase (AP), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured in serum and on the inner hepatocyte mitochondrial membrane (IHMM); the incidence of necrosis and the volume fractions of vessels, bile ducts and hepatocytes in the liver were also determined. HAL reduces the relative volumes of bile ducts, with no changes in levels of bilirubin and fractions, AP, ALT, AST and IHMM, but HAL associated with EHC reduces duct proliferation and the liver becomes more vulnerable to necrosis. In conclusion, the normal liver depends on HA flow and this dependence is more evident in the presence of EHC.

Hepatic morphology changes after hepatic artery ligation in extrahepatic cholestasis.

The present study was performed to correlate changes in hepatic morphology with hepatic artery ligation (HAL) in the presence of extrahepatic cholestasis (EHC). The study was conducted on 36 male Wistar rats weighing 350 to 400 g, divided at random into four groups of 9 animals each: group I, sham operation (control); group II, HAL; group III, bile duct ligation (BDL); group IV, HAL plus BDL. After seven days, liver fragments were obtained for morphological study. The relative volume of the bile ducts was I > II < III and III > IV (P < 0.05). These data indicate that arterial irrigation is important for the nutrition of the biliary tree. Seventy-six percent of the animals submitted to HAL plus BDL showed hepatic necrosis. In general, the liver probably becomes more dependent on HA flow in the presence of EHC.

Hepatic morphology changes after hepatic artery ligation in extrahepatic cholestasis

The present study was performed to correlate changes in hepatic morphology with hepatic artery ligation (HAL) in the presence of extrahepatic cholestasis (EHC). The study was conducted on 36 male Wistar rats weighing 350 to 400 g, divided at random into four groups of 9 animals each: group I, sham operation (control); group II, HAL; group III, bile duct ligation (BDL); group IV, HAL plus BDL. After seven days, liver fragments were obtained for morphiological study. The relative volume of the bile ducts was I>IIIV (P<0.05). These data indicate that arterial irrigation is important for the nutrition of the biliary tree. Seventy-six percent of the animals sumitted to HAL plus BDL showed hepatic necrosis. In general, the liver propably becomes more dependent on HA flow in the presence of EHC

Effect of hepatic artery ligation in rats with chronic extrahepatic cholestasis.

Because of the liver's dependence on arterial blood to exert its metabolic functions in cirrhosis of the liver, with or without thrombosis of the portal vein, the interruption of hepatic arterial flow for the palliative treatment of malignant tumors of the liver is counterindicated. However, the effects of arterial devascularization on the cholestatic liver are not fully understood. The objective of the present study was to investigate hepatic alterations due to hepatic artery ligation in rats with chronic extrahepatic cholestasis. Serum alkaline phosphatase, bilirubin and alanine aminotransferase were measured in rats 3 h after sham operation (group A, N = 29) or ligation of the hepatic artery (group B, N = 29). Alanine aminotransferase activity was significantly higher (P less than 0.05) in group B, demonstrating acute hepatocellular damage in animals with chronic extrahepatic cholestasis.

Effect of hepatic artery ligation in rats with chronic extrahepatic cholestasis

Because of the liver's dependence on arterial blood to exert metabolic functions in cirrhosis of the liver, this or without thrombosis of the portal vein, the interruption of hepatic arterial flow for the pallitive treatment of malignant tumors of the liver is counterindicated. However, the effects of arterial devascularization on the cholestatic liver are not fully understood. The objective of the present study was to investigate hepatic alterations due to hepatic artery ligation in rats with chroni extrahepatic cholestasis. Serum alkaline phosphatase, bilirubin and alanine aminotransferase were measured in rats 3 h after sham operation (group A, N = 29) or ligation of the hepatic artery (group B, N = 29). Alamine aminotransferase activity was significanty higher (P < 0.05) in group B, demonstrating acute hepatocellular damage in animals with chronic extrahepatic cholestasis