RESUMO
One of the main obstacles to studying the pathophysiology of lymphedema development is the lack of appropriate experimental models. Fol-lowing up on a mouse-tail method that has been described, we performed changes to the method which made it easier to perform in our hands and demonstrated similar results. Twenty C57Black mice were operated on using the previous tech-nique and euthanized after 3 or 6 weeks. Another twenty mice were submitted to the new technique developed in our laboratory and euthanized at the same time points. Tissue samples were collected from the proximal part of the tail (control) and from the distal part (lymphedema) for both mod-els. Animals in both operative groups developed marked edema in the distal part of the tail. This was characterized by lymph vessels dilation, edema, inflammatory cell infiltration, and adipose tissue deposition. Lymphedema was detected after 3 weeks in both models, reaching its maximum after 6 weeks. Adipocytes detected by histology (Oil red O staining) and molecular markers for adipogenesis, lymphangiogenesis and inflam-mation (lipin 1 and 2, SLP76, and F4-80) were demonstrated to be increased equally in both models. In conclusion, both models provide a reliable method to study lymphedema pathophys-iology. However, our modified technique is easier and faster to perform while still providing reliable and consistent results.
Assuntos
Adipogenia , Modelos Animais de Doenças , Inflamação/patologia , Linfangiogênese , Vasos Linfáticos/patologia , Cauda/patologia , Animais , Inflamação/cirurgia , Camundongos , Camundongos Endogâmicos C57BL , Cauda/cirurgiaRESUMO
Septic encephalopathy represents the most frequently observed form of encephalopathy in intensive care units. Interactions between the immune and nervous systems have been observed in experimental sepsis. Therefore, the aim of the current study was to characterize the effect of different severities of sepsis on encephalopathy and the inflammatory profile of the spleen. We hypothesized that different grades of sepsis severity would lead to variations in encephalopathy and activation of spleen cells. We induced sepsis of different severities in Balb/c mice by cecal ligature and puncture (CLP). Six and 12 h after CLP induction, behavioral impairment was assessed by the SmithKline/Harwell/Imperial College/Royal Hospital/Phenotype Assessment (SHIRPA) test. The animals were then killed, and the plasma, spleen, and hippocampus were removed. Levels of the encephalopathy marker S100ß were measured in plasma. Spleens were weighed and then a characterization of splenic lymphocytes was performed by flow cytometry (cytotoxic T lymphocyte, T helper lymphocytes, B lymphocytes, T regulatory cells, and Th17 cells). Cytokine levels in the spleen and hippocampus were determined by enzyme-linked immunosorbent assay (ELISA), and cytokine levels in plasma were performed with MilliPlex® technology. Our results showed that behavioral impairment as measured by the SHIRPA test and elevation in plasma S100ß levels were significant in moderate and severe CLP groups compared to those in the sham control group. Regarding immunological alterations, we were unable to observe changes in the weights of the spleen and the profile of lymphocytes 6 h after CLP. However, several cytokines, including IL-6, IL-10, and IL-1ß, were increased in spleen and plasma. In conclusion, we observed variations in encephalopathy as measured by plasma S100ß, which were mediated by the severity of sepsis; however, we did not observe a different activation of spleen cells 6 h post-CLP, despite evidence of inflammation. Taken together, our data indicate that the severity of sepsis impacts the brain in absence of a change in the spleen lymphocyte profile.
Assuntos
Encefalopatias/patologia , Ceco/patologia , Linfócitos/patologia , Punções , Sepse/etiologia , Baço/patologia , Animais , Comportamento Animal , Encefalopatias/sangue , Citocinas/metabolismo , Hipocampo/patologia , Ligadura , Masculino , Camundongos Endogâmicos BALB C , Tamanho do Órgão , Subunidade beta da Proteína Ligante de Cálcio S100/sangue , Sepse/sangue , Índice de Gravidade de Doença , Análise de SobrevidaRESUMO
Breast cancer is a prevalent neoplastic disease among women worldwide which treatments still present several side effects and resistance. Considering that cancer cells present derangements in their energetic homeostasis, and that peroxisome proliferator-activated receptor- gamma coactivator 1 (PGC-1) is crucial for cellular metabolism and redox signaling, the main objective of this study was to investigate whether there is a relationship between PGC-1 expression, the proliferation of breast cancer cells and the mechanisms involved. We initially assessed PGC-1ß expression in complementary DNA (cDNA) from breast tumor of patients bearing luminal A, luminal B, and HER2-overexpressed and triple negative tumors. Our data showed that PGC-1ß expression is increased in patients bearing HER2-overexpressing tumors as compared to others subtypes. Using quantitative PCR and immunoblotting, we showed that breast cancer cells with HER2-amplification (SKBR-3) have greater expression of PGC-1ß as compared to a non-tumorous breast cell (MCF-10A) and higher proliferation rate. PGC-1ß expression was knocked down with short interfering RNA in HER2-overexpressing cells, and cells decreased proliferation. In these PGC-1ß-inhibited cells, we found increased citrate synthase activity and no marked changes in mitochondrial respiration. Glycolytic pathway was decreased, characterized by lower intracellular lactate levels. In addition, after PGC-1ß knockdown, SKBR-3 cells showed increased reactive oxygen species production, no changes in antioxidant activity, and decreased expression of ERRα, a modulator of metabolism. In conclusion, we show an association of HER2-overexpression and PGC-1ß. PGC-1ß knockdown impairs HER2-overexpressing cells proliferation acting on ERRα signaling, metabolism, and redox balance.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Proteínas de Transporte/metabolismo , Regulação Neoplásica da Expressão Gênica , Genes erbB-2 , Redes e Vias Metabólicas , Oxirredução , Idoso , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular/genética , Feminino , Técnicas de Silenciamento de Genes , Inativação Gênica , Humanos , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA , Espécies Reativas de Oxigênio , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Carga Tumoral , Receptor ERRalfa Relacionado ao EstrogênioRESUMO
The auditory brainstem response (ABR) is a test widely used to assess the integrity of the brain stem. Although it is considered to be an auditory-evoked potential that is influenced by the physical characteristics of the stimulus, such as rate, polarity and type of stimulus, it may also be influenced by the change in several parameters. The use of anesthetics may adversely influence the value of the ABR wave latency. One of the anesthetics used for e ABR assessment, especially in animal research, is the ketamine/xylazine combination. Our objective was to determine the influence of the ketamine/xylazine anesthetic on the ABR latency values in adult gerbils. The ABRs of 12 adult gerbils injected with the anesthetic were collected on three consecutive days, or a total of six collections, namely: pre-collection and A, B, C, D, and E collections. Before each collection the gerbil was injected with a dose of ketamine (100 mg/kg)/xylazine (4 mg/kg). For the capture of the ABR, 2000 click stimuli were used with rarefaction polarity and 13 stimuli per second, 80 dBnHL intensity and in-ear phones. A statistically significant difference was observed in the latency of the V wave in the ABR of gerbils in the C and D collections compared to the pre-, A and E collections, and no difference was observed between the pre-, A, B, and E collections. We conclude that the use of ketamine/xylazine increases the latency of the V wave of the ABR after several doses injected into adult gerbils; thus clinicians should consider the use of this substance in the assessment of ABR.
Assuntos
Animais , Masculino , Anestésicos/farmacologia , Potenciais Evocados Auditivos do Tronco Encefálico/efeitos dos fármacos , Ketamina/farmacologia , Xilazina/farmacologia , Anestésicos/administração & dosagem , Limiar Auditivo/efeitos dos fármacos , Gerbillinae , Ketamina/administração & dosagem , Tempo de Reação , Xilazina/administração & dosagemRESUMO
The auditory brainstem response (ABR) is a test widely used to assess the integrity of the brain stem. Although it is considered to be an auditory-evoked potential that is influenced by the physical characteristics of the stimulus, such as rate, polarity and type of stimulus, it may also be influenced by the change in several parameters. The use of anesthetics may adversely influence the value of the ABR wave latency. One of the anesthetics used for e ABR assessment, especially in animal research, is the ketamine/xylazine combination. Our objective was to determine the influence of the ketamine/xylazine anesthetic on the ABR latency values in adult gerbils. The ABRs of 12 adult gerbils injected with the anesthetic were collected on three consecutive days, or a total of six collections, namely: pre-collection and A, B, C, D, and E collections. Before each collection the gerbil was injected with a dose of ketamine (100 mg/kg)/xylazine (4 mg/kg). For the capture of the ABR, 2000 click stimuli were used with rarefaction polarity and 13 stimuli per second, 80 dBnHL intensity and in-ear phones. A statistically significant difference was observed in the latency of the V wave in the ABR of gerbils in the C and D collections compared to the pre-, A and E collections, and no difference was observed between the pre-, A, B, and E collections. We conclude that the use of ketamine/xylazine increases the latency of the V wave of the ABR after several doses injected into adult gerbils; thus clinicians should consider the use of this substance in the assessment of ABR.
Assuntos
Anestésicos/farmacologia , Potenciais Evocados Auditivos do Tronco Encefálico/efeitos dos fármacos , Ketamina/farmacologia , Xilazina/farmacologia , Anestésicos/administração & dosagem , Animais , Limiar Auditivo/efeitos dos fármacos , Gerbillinae , Ketamina/administração & dosagem , Masculino , Tempo de Reação , Xilazina/administração & dosagemRESUMO
Sepsis is a systemic inflammatory response that can lead to tissue damage and death. In order to increase our understanding of sepsis, experimental models are needed that produce relevant immune and inflammatory responses during a septic event. We describe a lipopolysaccharide tolerance mouse model to characterize the cellular and molecular alterations of immune cells during sepsis. The model presents a typical lipopolysaccharide tolerance pattern in which tolerance is related to decreased production and secretion of cytokines after a subsequent exposure to a lethal dose of lipopolysaccharide. The initial lipopolysaccharide exposure also altered the expression patterns of cytokines and was followed by an 8- and a 1.5-fold increase in the T helper 1 and 2 cell subpopulations. Behavioral data indicate a decrease in spontaneous activity and an increase in body temperature following exposure to lipopolysaccharide. In contrast, tolerant animals maintained production of reactive oxygen species and nitric oxide when terminally challenged by cecal ligation and puncture (CLP). Survival study after CLP showed protection in tolerant compared to naive animals. Spleen mass increased in tolerant animals followed by increases of B lymphocytes and subpopulation Th1 cells. An increase in the number of stem cells was found in spleen and bone marrow. We also showed that administration of spleen or bone marrow cells from tolerant to naive animals transfers the acquired resistance status. In conclusion, lipopolysaccharide tolerance is a natural reprogramming of the immune system that increases the number of immune cells, particularly T helper 1 cells, and does not reduce oxidative stress.
Assuntos
Animais , Masculino , Camundongos , Citocinas/imunologia , Modelos Animais de Doenças , Lipopolissacarídeos/imunologia , Estresse Oxidativo/imunologia , Sepse/imunologia , Proliferação de Células , Tolerância Imunológica/imunologia , Camundongos Endogâmicos BALB CRESUMO
Sepsis is a systemic inflammatory response that can lead to tissue damage and death. In order to increase our understanding of sepsis, experimental models are needed that produce relevant immune and inflammatory responses during a septic event. We describe a lipopolysaccharide tolerance mouse model to characterize the cellular and molecular alterations of immune cells during sepsis. The model presents a typical lipopolysaccharide tolerance pattern in which tolerance is related to decreased production and secretion of cytokines after a subsequent exposure to a lethal dose of lipopolysaccharide. The initial lipopolysaccharide exposure also altered the expression patterns of cytokines and was followed by an 8- and a 1.5-fold increase in the T helper 1 and 2 cell subpopulations. Behavioral data indicate a decrease in spontaneous activity and an increase in body temperature following exposure to lipopolysaccharide. In contrast, tolerant animals maintained production of reactive oxygen species and nitric oxide when terminally challenged by cecal ligation and puncture (CLP). Survival study after CLP showed protection in tolerant compared to naive animals. Spleen mass increased in tolerant animals followed by increases of B lymphocytes and subpopulation Th1 cells. An increase in the number of stem cells was found in spleen and bone marrow. We also showed that administration of spleen or bone marrow cells from tolerant to naive animals transfers the acquired resistance status. In conclusion, lipopolysaccharide tolerance is a natural reprogramming of the immune system that increases the number of immune cells, particularly T helper 1 cells, and does not reduce oxidative stress.
Assuntos
Citocinas/imunologia , Modelos Animais de Doenças , Lipopolissacarídeos/imunologia , Estresse Oxidativo/imunologia , Sepse/imunologia , Animais , Proliferação de Células , Tolerância Imunológica/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
We conducted karyological studies on one colony consisting of 12 Colombian and 34 Bolivian squirrel monkeys and a second colony of 47 monkeys imported into the United States between 1968 and 1974 through six importers. All animals in the first colony showed six acrocentric pairs of chromosomes. Bolivian monkeys were phenotypically distinguishable by their large size and coloration. In the second colony, 19 Peruvian, seven Colombian, five Bolivian and six Guyanan monkeys were correctly identified phenotypically and had five, six, six, and seven acrocentric pairs of chromosomes, respectively. Among Peruvian monkeys, 34.5% of the phenotypic classifications were in error.