A técnica de mobilização neural (NM) clinicamente é eficaz, porém ainda é pouco fundamentada. Neste trabalho, submetemos ratos Wistar no 14º dia após a lesão constritiva crônica (CCI) do nervo isquiático ao tratamento com NM, em 10 sessões, e avaliamos o comportamento doloroso utilizando testes comportamentais para hiperalgesia e alodinia. Ainda, observamos através de ensaios de Western blotting o envolvimento das células gliais e do fator neurotrófico derivado do cérebro (BDNF). No estudo comportamental, os animais com CCI mostraram diminuição no limiar nociceptivo, tratados com a NM apresentaram melhora no comportamento doloroso. Os ensaios de Western blotting mostraram que após a CCI houve aumento de OX-42, GFAP e BDNF, na medula, tálamo e mesencéfalo, também observado em analise de imuno-histoquímica e após a NM observamos diminuição desses mediadores através da primeira técnica mencionada. Sendo assim, sugerimos que a técnica de NM é eficaz como terapia analgésica, sendo possível observar o envolvimento das células gliais e do BDNF neste modelo experimental. / The technique of neural mobilization (NM) is clinically effective, although it is still poorly reasoned. In this study, Wistar rats on day 14th after chronic constrictive injury (CCI) of the sciatic nerve were submitted to treatment with NM in 10 sessions, and it was evaluated the painful behavior using tests for hyperalgesia and allodynia. Also, we observed through Western blotting assays the involvementof glial cells and brain-derived neurotrophic factor (BDNF). In the behavioral study, animals with CCI showed a decrease in nociceptive threshold, and those treated with NM showed an improvement in pain behavior. Western blotting assays showed an increase after CCI of OX-42, GFAP and BDNF levels in the spinal cord, thalamus and midbrain, also observed in immunohistochemical analysis, and after the NM we observed a decrease of these mediators through the first technique mentioned. Therefore, we suggest that the NM technique is an effective analgesic therapy, and it is possible to observe the involvement of glial cells and BDNF in this experimental model.

O estudo avaliou o óleo-resina (OR) proveniente da Copaifera langsdorffii quanto: à citotoxicidade; ao potencial cicatrizante em modelos animais; aos efeitos no modelo ex vivo de cicatrização da pele humana; à atividade antimicrobiana in vitro e in vivo. A citotoxicidade do OR (1, 10, 50, 100, 500 e 1000 µg/mL) foi avaliada em culturas de fibroblastos 3T3 pela determinação da viabilidade e proliferação celular. A atividade cicatrizante foi avaliada em úlceras cutâneas em orelha de coelho, com aplicações tópicas diárias, por 21 dias, do OR in natura e em creme (10 e 25%) e controles [creme base (Cr) e solução fisiológica]; no dorso de ratos, o tratamento consistiu de creme a 10% e os mesmos controles, por 14 dias. As úlceras foram avaliadas por análise de imagem, índices de cicatrização e histologia nos dias 2, 7, 14 e 21. Parâmetros bioquímicos (hidroxiprolina e metaloproteinases) foram incluídos na investigação da cicatrização em ratos. No modelo ex vivo, os fragmentos de pele receberam diariamente: OR in natura, cremes a 10 e 25% e controles (Cr e meio de cultura (DMEM), por 21 dias, e foram avaliados nos dias 0, 1, 3, 7, 14 e 21, por análises clínico-fotográfica e histológica. A atividade antimicrobiana in vitro foi avaliada pela determinação das concentrações inibitória e bactericida mínimas para bactérias Gram-positivas: S. aureus (ATCC 6538), S. pyogenes (ATCC 19615) e Enterococcus faecalis (ATCC 10541) e Gram-negativas: P. aeruginosa (ATCC 2327) e E. coli (ATCC 10538). A atividade antimicrobiana in vivo foi avaliada nas úlceras infectadas com S. aureus ou S. pyogenes em ratos, pela contagem bacteriana no exsudato e biópsias nos dias 2, 7 e 14 após úlceras e inoculação bacteriana além da atividade cicatrizante. O OR não apresentou toxicidade aos fibroblastos até 100 µg/mL, com viabilidade e proliferação de 90 a 100%. Promoveu melhor cicatrização a 10%, nos modelos orelha de coelho e dorso de ratos, enquanto, in natura ocasionou agressão tecidual. O modelo ex vivo manteve-se viável por 21 dias; a 10% o OR inibiu a reepitelização das úlceras, diferente do controle (DMEM), evidenciando que sua atividade cicatrizante é dependente do processo inflamatório, ausente neste modelo. Apresentou atividade antimicrobiana in vitro (S. aureus, S. pyogenes e E. faecalis), a qual se manteve in vivo observada pelo estímulo cicatrizante do OR a 10% em creme nas úlceras infectadas com S. aureus ou S. pyogenes em ratos. Os resultados evidenciaram que o OR da C. langsdorffii não foi citotóxico, apresentou atividade antimicrobiana in vitro e in vivo, além de importante atividade cicatrizante dependente, essencialmente da fase inflamatória, demonstrada nos modelos animais e ausente no modelo ex vivo, contribuindo para o conhecimento quanto ao seu mecanismo de ação, eficácia e segurança para o uso clínico na cicatrização de úlceras cutâneas. / The study evaluated oleoresin (OR) obtained from Copaifera langsdorffii regarding its: cytotoxicity; wound healing potential in animal models; effects on the ex vivo model of human skin healing; and antimicrobial activity in vitro and in vivo. The cytotoxic effect of OR (1, 10, 50, 100, 500 and 1000 µg/ mL) was assessed on 3T3 fibroblasts by viability and proliferation assays. The wound healing activity was performed in a rabbit ear model, with daily topical treatment, over 21 days: in natura OR, and at 10 and 25% cream, and controls [base cream (Cr) and saline]; in dorsal wounds in rats, the treatment consisted of daily topical application of 10% OR cream and the same controls, over 14 days. The wounds were assessed by image analysis, wound healing rates and histology on days 2, 7 and 14. Biochemical tests (hydroxyproline and metalloproteinases) were included for the healing investigation in rats. In the ex vivo model, skin fragments received daily: in natura OR, 10 and 25% creams, and controls (Cr and culture medium (DMEM), for 21 days, and on days 0, 1, 3, 7, 14 and 21, fragments were analyzed by photographical and histological analyses. The in vitro antimicrobial activity was assessed by the minimal inhibitory and bactericidal concentrations assays, on Gram-positive: S. aureus (ATCC 6538), S. pyogenes (ATCC 19615) and Enterococcus faecalis (ATCC 10541) and Gram-negative: P. aeruginosa (ATCC 2327) and E. coli (ATCC 10538). The in vivo antimicrobial activity was assessed in wounds infected either with S. aureus or S. pyogenes, by bacterial count in the exudates and biopsies on days 2, 7 and 14 after wounding and bacterial inoculation, along with the healing activity. OR was not cytotoxic to fibroblasts up to 100 µg/mL, showing 90 to 100% cell viability and proliferation. OR 10% cream enhanced healing in rabbits and in rats, however, in natura it caused aggression to the tissue. The ex vivo model was viable for 21 days; at 10% OR inhibited wound reepithelialization, different from the control (DMEM), showing evidence that healing properties of OR depend on the inflammatory phase, absent in this model. OR showed in vitro antimicrobial activity (S. aureus, S. pyogenes and E. faecalis), which was maintained in vivo, observed by a healing stimulus of 10% OR cream in wounds infected with S. aureus or S. pyogenes, in rats. The results showed that OR from C. langsdorffii showed no cytotoxicity, and presented in vitro and in vivo antimicrobial activity. It also enhanced healing, depending, essentially, on the inflammatory phase, demonstrated by the animal models of injury and repair and absent in the ex vivo model. These findings contribute to an understanding of the OR mechanism of action, efficacy and safety for clinical use in wound healing.

Os recém-nascidos pré-termo extremos, por estarem ainda em uma fase do desenvolvimento pulmonar anterior à alveolização, estão mais vulneráveis a fatores como a hiperoxia, o barotrauma, o volutrauma e, o uso de medicações como os corticosteróides. A ação destes fatores, associada à imaturidade pulmonar, pode alterar a organização estrutural pulmonar, interferindo na alveolização e na organização de fibras elásticas e de colágeno. Estas alterações são comuns à \"nova\" displasia broncopulmonar, patologia freqüente entre os recém-nascidos pré-termo, especialmente os extremos. Criou-se um modelo experimental de restrição nutricional e hiperoxia, em coelhos pré-termo, com o objetivo de analisar os efeitos da restrição nutricional e da hiperoxia sobre a arquitetura pulmonar (número de alvéolos, intercepto linear médio(ILm), área de superfície interna(ASI), espessura de septo interalveolar), especialmente em relação à deposição de fibras elásticas e de colágeno. Após a realização de cesárea, em coelhas New Zealand White, com idade gestacional de 28 dias, seus filhotes foram divididos em 4 grupos, de acordo com a dieta e a concentração de oxigênio administradas: GIA (dieta padrão e FiO2=0,21) (n=120); GIB (dieta padrão e FiO2>0,95) (n=232); GIIA (restrição nutricional e FiO2=0,21) (n=72); GIIB (restrição nutricional e FiO2>0,95) (n=368). A restrição nutricional foi definida como uma redução em 30% de todos os nutrientes, em relação à dieta padrão. Os coelhos foram pesados, diariamente. Após o sacrifício, aos 7 e 11 dias de vida, os pulmões foram removidos, pesados e fixados em formol tamponado 10%, com uma pressão de 30 cm de H2O. O volume pulmonar total foi medido pelo método de deslocamento de água. Nos cortes histológicos utilizou-se as seguintes colorações: hematoxilina-eosina, para contagem do número de alvéolos, ILm, ASI, medida de septo interalveolar; orceína-resorcina modificada para análise de fibras elásticas e picrosirius, para análise de colágeno. Os resultados foram apresentados através de médias e realizou-se ANOVA para comparação dos resultados, considerando-se um p < 0,05 como significante. A restrição nutricional alterou o crescimento somático, reduzindo o ganho de peso e, alterou o crescimento e a morfologia pulmonares, reduzindo o volume pulmonar, o número de alvéolos e a deposição de fibras elásticas e de colágeno aos 7 e 11 dias de vida. A hiperoxia elevou a mortalidade e modificou a arquitetura pulmonar, reduzindo o número de alvéolos e aumentando suas dimensões, bem como aumento da espessura dos septos interalveolares, aos 7 e 11 dias de vida. Aos 11 dias, a hiperoxia reduziu a deposição de colágeno. A restrição nutricional associada à hiperoxia teve os seus efeitos intensificados sobre a redução do número de alvéolos e do depósito de colágeno, aos 7 e 11 dias. Estes resultados ilustram o papel da nutrição como modulador da lesão pulmonar pela hiperoxia, em pulmões em desenvolvimento / Characteristic pathologic findings in bronchopulmonary dysplasia are the presence of alveolization impairment, widened alveolar septa, disordered collagen and elastic fibers deposition. There are likely to be multiple factors that result in the histological changes seen with the development of bronchopulmonary dysplasia, besides the lung imaturity: hyperoxia, volutrauma, barotrauma, nutrition. The aim of this study is to developed an experimental model, in preterm rabbits, to evaluate the effects of nutritional restriction and hyperoxia on lung weight and volume, alveoli number, mean linear intercept (Lm), internal surface area (ISA), alveolar septal thickness, elastic fibers and collagen density. After c-section, 28 days preterm New Zealand White rabbits were randomized into 4 groups: GIA (regular diet and FiO2=0,21) (n=120); GIB (regular diet and FiO2>0,95) (n=232); GIIA (nutritional restriction and FiO2=0,21) (n=72); GIIB (nutritional restriction and FiO2>0,95) (n=368). They were kept in incubators, warmer and humidified oxygen (Plexiglas chamber). Nutritional restriction was defined as caloric-proteic intake limited to 70% of the regular diet. After sacrifice with 7 and 11 days, the lungs were removed and fixed in 10% formalin under 30 cm H2O transtracheal pressure. The total lung volume was measured by displacement water. Lung slices were stained with hematoxylin and eosin for alveoli number, Lm, ISA and alveolar septal thickness; resorcin-orcein for elastic fibers and Picrosirius for collagen (fibers/parenchyma pointy counting). Statistical analysis was done by ANOVA. Level of significance was set at 0,05. Values are means +- sd. It was observed that GIA and GIB (regular diet) put on more weight than nutritional restriction groups from 8th till 11th day of life. Nutritional restriction influenced the ponderal evolution and reduced lung volume, alveoli number, collagen and elastic fibers deposition at 7th and 11th days of life. Hyperoxia elevated the mortality. In relation to lung architetural, the hyperoxia reduced alveoli number and elevated Lm, besides widened alveolar septal at 7th and 11th days of life. At 11th days, hyperoxia reduced collagen deposition, too. Nutritional restriction and hyperoxia had an additive efect on the reduction of alveoli number and collagen deposition. These findings suggest that nutrition has a modulator effect on lung injury by hyperoxia. This study strengthened the contribution of nutrition on preterm lung development

Introdução: Há crescentes evidências que sinalizam o papel do estresse crônico como desencadeante e mesmo perpetuador de crises asmáticas, bem como a importância da produção de muco e do transporte mucociliar na fisiopatologia da asma brônquica. Objetivos: Deste modo, consideramos relevante avaliar em cobaias com inflamação alérgica crônica pulmonar como o estresse físico repetido, induzido pela natação forçada, modula as respostas de transporte mucociliar, as propriedades do muco, o infiltrado eosinofílico e a expressão de IL-13 no epitélio e nas células inflamatórias na parede das vias aéreas. Métodos: Os animais receberam inalações duas vezes por semana durante quatro semanas com doses crescentes de ovoalbumina (grupo OVA e OVA-NAT) ou solução fisiológica (grupo SAL e SAL-NAT). Após 24 horas da quarta inalação os animais dos grupos denominados SAL-NAT e OVA-NAT foram submetidos ao protocolo de natação forçada por dez dias com intervalo de dois dias, para a indução do estresse. Após 72 horas da última inalação, os animais foram anestesiados, sendo testadas: a velocidade de transporte mucociliar (VTM), a freqüência de batimento ciliar (FBC), e a diferença de potencial transepitelial (PD). Após estas medidas foram coletadas amostras de muco para avaliação do ângulo de contato (AC) e da transportabilidade pela tosse (TT). Foi coletada uma amostra de sangue para a dosagem de cortisol sérico. Os pulmões foram retirados, fixados e submetidos à coloração de LUNA, para identificação de eosinófilos, e à técnica imunohistoquímica para detecção da expressão de IL-13 no epitélio brônquico e nas células inflamatórias presentes na parede das vias aéreas. As adrenais também foram retiradas, imediatamente pesadas e submetidas à coloração de hematoxilina e eosina para avaliação histopatológica, utilizando a morfometria para determinação das áreas de cada uma de suas camadas. Resultados: Os Resumo animais do grupo OVA apresentaram uma redução da VTM, do PD, da TT e aumento do AC, da área de epitélio de vias aéreas, de muco ácido, do número de eosinófilos e da expressão de IL-13, no epitélio brônquico e nas células inflamatórias presentes ao redor das vias aéreas, comparativamente aos controles (p<0,05 para todas as comparações). Houve redução da área total da adrenal e de cada uma de suas camadas comparativamente aos controles (p<0,05 para todas as comparações). Os animais submetidos ao protocolo de estresse (grupos SAL-NAT e OVA-NAT) apresentaram aumento nos níveis de cortisol sérico (p<0,05), no peso das adrenais comparativamente aos grupos e OVA (p<0,05). Os animais submetidos ao protocolo de estresse e expostos à ovoalbumina (grupo OVA-NAT) mostraram redução na VTM, aumento no AC, na área de muco ácido do epitélio brônquico e em todas as camadas das adrenais, comparativamente aos resultados obtidos nos animais do grupo OVA (p<0,05 para todas as comparações). Conclusão: O processo inflamatório crônico pulmonar altera o transporte mucociliar e as propriedades reológicas do muco, o que se associou ao recrutamento eosinofílico, aumento da expressão de IL-13 no epitélio brônquico e nas células inflamatórias presentes na parede brônquica. Além disto, observamos a redução de todas as camadas da adrenal. Neste modelo experimental, o estresse físico repetido reduz o transporte mucociliar devido a alterações das propriedades do muco, particularmente potencializando o aumento de muco ácido e de sua hidrofobicidade e adesividade. Estes efeitos parecem estar relacionados à ativação adrenal, mas independentes do recrutamento eosinofílico e da resposta Th2 mediada pela IL-13 / Background: It has increasing evidence linking the role of stress in the onset of asthma exacerbation and in the maintenance of asthmatic crises, as well as the importance of mucus production and the mucociliary clearance in the asthma physiopathology. Objectives: So, we consider relevant to evaluate in guinea pigs with pulmonary chronic allergic inflammation how the induced repeated physical stress, caused by forced swimming, modulates the mucociliary clearance, the mucus properties, the eosinophilic infiltration and the IL-13 expression on bronchial epithelial cells in the airway wall. Methods: The animals had received inhalations two times per week during four weeks with increasing doses of ovalbumin (OVA and OVA-S groups) or saline solution (SAL and SAL-S groups). After twenty four hours of the 4th inhalation, the animals (named as SAL-S and OVA-S groups) had been submitted to the protocol of forced swimming, per ten days with an interval of two days, for the stress induction. After 72 hours of the last inhalation, the animals were anaesthetized and the tracheal mucus clearance (TMC), the ciliary beat frequency (CBF), and the difference of transepithelial potential difference (PD) were measured. After these measurements had been done mucus samples were collected for evaluation of the contact angle (CA) and cough transportability (CT). To serum cortisol dosage a little amount of blood was collected. The lungs were dissected, fixed and submitted to histological techniques, like LUNA stain for identification of eosinophils and the IL-13 immunohistochemistry technique for its detection in the bronchial epithelium and in the inflammatory cells present in the airways walls. The adrenal glands were excised, immediately weighed and submitted to haematoxylin and eosin stain for histopathological evaluation using the morphometry to determine the areas of each of their layers. Results: The animals of OVA Summary group presented a reduction of the TMC, the PD, and the CT. On the other hand they presented an increase of the CA, of the airways epithelial area, of the acid mucus, of the eosinophils number as well as on the IL-13 expression in the airways epithelium and on the inflammatory cells around the airways, compared to the controls (p<0.05 for all comparisons). There was a reduction of the adrenal gland total area and on each one of its layers compared to the controls (p<0.05 for all comparisons). The animals submitted to the forced swim stress protocol (SAL-S and OVA-S groups) showed an increase on serum cortisol levels, on adrenal gland weight compared to OVA group (p<0.05). The animals submitted to the forced swim stress protocol and to the sensitization protocol with ovalbumin (OVA-S) showed a reduction in the TMC and an increase on the CA, bronchial epithelial acid mucus area and in all adrenal gland layers compared to the results obtained in the OVA group animals (p<0.05 for all comparisons). Conclusions: The chronic inflammatory pulmonary process alters the mucociliary transport and mucus rheological properties which was associated to eosinophilic recruitment, increases in the IL-13 expression on bronchial epithelial cells and inflammatory cells in airway walls. In addition, we observed a reduction in all adrenal zones. In this experimental model the repeated physical stress reduces the mucociliary clearance due to the mucus rheological properties alterations, particularly increasing the amount of acid mucus and its wettability and adhesivity. These effects seem to be related to the adrenal activation but independently of the eosinophilic recruitment and of Th2 responses mediated by IL-13

O objetivo do presente trabalho foi propiciar o acesso in vivo ao epitélio respiratório e estudar a frequência de batimento ciliar (FBC) e a diferença de potencial transepitelial (DP) em um modelo murino de doença alérgica das vias aéreas induzida por ovoalbumina. Camundongos Swiss foram sensibilizados com ovoalbumina (OVA) através de duas injeções intraperitoneais de alérgico com o adjuvante hidróxido de alumínio (dias 0 e 14) e quatro inalações de OA 1% (dias 22, 24, 26 e 28). O grupo controle (S) foi tratado com salina 0,9 % seguindo o mesmo protocolo. Após 48h da última inalação, os camundongos foram anestesiados, a traquéia foi exposta longitudinalmente (1x4 mm) e o epitélio pode ser visualizado. A FBC foi mensurada pela técnica estroboscópica antes (basal) e logo após a administração inalatória das drogas (salbutamol e brometo de ipratrópio). A DP foi mensurada nos grupos S e OVA. Foram avaliados o lavado broncoalveolar e o remodelamento do epitélio da cavidade nasal, traquéia e vias aéreas distais. Nenhuma diferença foi encontrada na FBC basal entre os grupos (OVA e S), no entanto o grupo OVA mostrou uma DP basal significativamente menor. A inalação de salbutamol (3.5.10-3M ou 3.5.10-4M) elevou a FBC nos grupos estudados (p<0,05). O brometo de ipratrópio (10- 4M e 6.10-4M) não influenciou a FBC basal. Nossos resultados mostraram que é possível avaliar a FBC e a DP in vivo em um modelo murino de doença pulmonar alérgica crônica, e indicam que o processo inflamatório não afeta a FBC, mas contribui para o aumento de muco nas vias aéreas com conseqüências deletérias ao transporte mucociliar facilitando a retenção / The aim of the present work was to propitiate the in vivo assessment of the respiratory epithelium. The effects of salbutamol and ipratropium bromide on ciliary beat frequency (CBF) in a murine model of allergic airway disease were addressed. Transepithelial electric potential difference (PD) was also measured in order to verify the integrity of the epithelial barrier. Mice were sensitized with ovalbumin (OVA) by two intraperitoneal injections of allergen (days 0 and 14) and four inhalations of OVA 1% (days 22, 24, 26 and 28). The control group was treated with saline following the same procedures. After 48 hs of the last inhalation, mice were anesthetized, trachea was opened longitudinally (1 x 4 mm) and the ciliated epithelium could be visualized. CBF was measured by a modification on the videoscopic technique. We measured the CBF before and just after the administration of aerosolized substances. The PD was also measured on groups OVA and S. Additionally, the eosinophil cell count was measured on broncoalveolar lavage (BAL) in order to access the magnitude of airway inflammation. No difference on baseline CBF was noticed between groups (OVA and S), however the OVA group had a significantly lower PD. The administration of aerosolized capsaicin (3.10-9M) and salbutamol (3.5.10-3M or 3.5.10-4M) increased CBF in all groups studied. Ipratropium bromide (10-4M and 6.10- 4M) did not influence the CBF. The eosinophil cell count in broncoalveolar lavage was higher in OVA group compared to S group. CBF and PD results indicate that the inflammatory process does not affect the ciliary beat frequency but augments the amount of mucus in the airway, with deleterious consequences to the mucociliary transport facilitating mucus retention. Our results demonstrated for the first time the possibiliy of studying airway epithelium in an in vivo murine model of allergic airway disease

Muitos estudos de câncer têm sido desenvolvidos, mas os mecanismos moleculares da tumorigênese e a resposta imune contra tumores não foi completamente elucidada. RMAS é uma linhagem celular mutante derivada de RMA. Ao contrário da última, RMAS é deficiente de MHC I e, portanto, é avlvo de células NK. O objetivo deste trabalho foi o uso deste par de células para estabelecer modelos murinos que possam ser usados para entender a resposta imune entre células CD8 e NK contra tumor e investigar o efeito da expressão de moléculas antiapoptóticas no comportamento tumoral in vivo. Essa abordagem pode prover informações relevantes para o desenvolvimento de novas terapias. Para desenvolver células EGFP, foi usado um vetor retroviral bicistrônico contendo o gene Egfp. Para desenvolver os modelos experimentais, camundongos C57BL6 WT foram injetados iv com diferentes números de células e curvas de sobrevivência foram geradas. Os padrões de doença e infiltração tumoral foram observadas por análises macroscópica, microscópica e por detecção de EGFP em tecidos. In vivo, células RMA. induziram paralisia enquanto RMA-S.Egfp, ascite. RMA.Egfp infiltrou a medula óssea enquanto RMA-S.Egfp, tecidos diferentes como fígado, rins e peritôneo, mas não a medula óssea. Os sinais clínicos apareceram após 15 dias da inoculação de >104 células e a morte, em 30 dias. Números <103 células não induziram doença nem morte, mas protegeram de ambas quando re-inoculadas 106 células RMA.Egfp. Camundongos CD4KO e CD8KO paralisaram e morreram antes do que os WT. Células RMA.BclW.Egfp foram mais resistentes a apoptose do que células RMA.Egfp in vitro e provocaram características clínicas piores: paralisia e morte anteriores, inchaço de membros e hemorragia de fígado e rins. / Many cancer studies have been developed, however the molecular mechanisms of tumorigenesis and immune responses to tumor is not completely elucidated. RMAS cells are a mutant lymphoma line derived from RMA cells. In contrast to the latter, RMAS are deficient in MHCI and, therefore, are targets for NK cells. Our aims were use these pair of cells to establish mouse models that can be used to understand CD8T vs NK cell immune responses to tumors and investigate the effect of expression of antiapoptotic molecules in tumor behavior in vivo. The combination of these approaches should provide relevant information for the development of novel immunotherapy. To develop EGFP cells we used a bicistronic retroviral vector containing Egfp gene. To develop the experimental models, WT C57BL/6 mice were iv injected with different cell numbers and survival curves were produced. In addition, clinical features and tumor spread was observed by macroscopy, microscopy and EGFP detection of tumor cell analysis in tissues. When injected in vivo, RMA.Egfp cells induced progressive paralysis while RMA-S.Egfp promoted ascites. RMA.Egfp cells infiltrated the bone marrow, while RMA-S.Egfp were found in different tissues such as liver, kidney and the peritoneum cavity, but were not found in bone marrow. The symptoms appeared 15 days post injection of >104 cells and the death was 30 days. Numbers of <103 cells do not induced pathology or death, but protected to paralysis and death when re-injected 106 RMA.Egfp cells. CD4KO and CD8KO mice showed paralysis and death earlier than WT mice. RMA.BclW.Egfp cells were more resistant to apoptosis than RMA.Egfp in vitro and induced worse clinical features in vivo: earlier paralysis and death, swelling of members, haemorragia of liver and kidneys.

O transplante (Tx) de ilhotas pancreáticas (IP) é uma atraente alternativa para o tratamento do diabetes melito tipo 1. No entanto, para evitar a rejeição há necessidade de imunossupressão. Uma nova idéia de tolerância surge a partir do paradoxo imunológico, onde a mãe, imunologicamente competente, não rejeita o embrião durante a gravidez. Uma das hipóteses é que células da placenta expressam a molécula IDO, a qual protege o embrião do ataque imunológico materno. O objetivo do estudo foi analisar o efeito da indução da expressão da IDO em IP em transplante experimental de IP. Para tanto, as seguintes etapas de padronização foram necessárias. Etapa 1: Padronização da perfusão e digestão do tecido pancreático de rato e determinação do método para a purificação das IP, comparando-se diferentes gradientes de densidade: descontínuo de Ficoll, contínuo de Ficoll e contínuo de iodixanol. Foi demonstrado que o gradiente contínuo de iodixanol fornece maior pureza e maior número de IP íntegras e funcionais. Etapa 2: Padronização do Tx experimental de IP sob a cápsula renal para avaliação do número mínimo de IP transplantadas para reverter o diabetes induzido por estreptozotocina, definido como glicemia >300mg/Kg. Foram transplantadas entre 200 a 3.000 IP por experimento. A rejeição das IP foi analisada pela sobrevida das IP (permanência da glicemia <300mg/dL), tanto em Tx isogênico (Lewis-Lewis) como em alogênico (Sprague-Dawley-Lewis). Para reverter o diabetes foram necessárias no mínimo 2.500 IP. No transplante entre ratos isogênicos (n=6) não houve rejeição das IP. Já no transplante entre animais alogênicos (n=12), as IP apresentaram uma curta sobrevida pós-Tx (11±1 dias; p<0,01 vs. Tx isogênico). Dez dias pós-Tx, houve um grande infiltrado de macrófagos e linfócitos T no enxerto alogênico e uma diminuição significativa da expressão de insulina (p<0,001 vs. Tx isogênico). Etapa 3: Construção do vetor de expressão para IDO. A partir de RNA extraído de placenta de rata no 10º dia de gestação, foi amplificada a seqüência completa do cDNA para IDO, utilizando-se RT-PCR. Em seguida, o cDNA para IDO foi inserido em vetor de expressão (vetor-IDO). Etapa 4: Transfecção do vetor-IDO nas IP. O vetor-IDO foi introduzido nas IP através de lipofecção (Lipofectamina 2000), testando-se diferentes concentrações do vetor-IDO (0, 0,5, 1 e 10 ng/uL) e diferentes períodos de incubação (1h, 15h e 24h). A expressão de IDO nas IP foi confirmada por RT-PCR e imuno-histoquímica. A incubação com 10 ng/uL de vetor-IDO durante 24h foi eficaz para induzir a expressão de IDO nas IP, confirmada a nível de RNAm (RT-PCR) e de proteína (imuno-histoquímica). A eficiência da transfecção em nível funcional foi confirmada pela degradação de triptofano em cultura (dosagem de triptofano por HPLC). Etapa 5: Onze transplantes alogênicos (Sprague-Dawley-Lewis) com IP transfectadas com vetor-IDO foram realizados para analisar o efeito da IDO. Três animais foram sacrificados para análise de imuno-histoquímica e 8 animais foram acompanhados por 45 dias. A sobrevida das IP transfectadas com vetor-IDO foi significativamente maior comparada com a sobrevida de IP não-transfectadas (p<0,01). O estudo conclui que a expressão da IDO protege as IP aumentando a sobrevida das IP. / Transplantation (Tx) of pancreatic islets (PI) is an attractive alternative of treatment for type 1 diabetes mellitus. However, continuous immunossupression is necessary in order to avoid allograft rejection. A new idea of tolerance is based on the immunological paradox, during pregnancy, in that the mother, immunologically competent, does not reject the semi-allogeneic fetus. The hypothesis is that the placenta produces IDO molecules, which protect the embryos against the maternal immunologic attack. The aim of this study was to analyze the effect of the induction of the IDO expression into PI in an experimental model of PI transplantation. The following steps for standardization were necessary. Step 1: Besides the standardization of the rat pancreas perfusion and digestion, the best method for purification of the PI was determined, comparing several density gradients: Ficoll discontinuous, Ficoll continuous and iodixanol continuous. The iodixanol continuous gradient was able to provide high purity and a high number of intact and functional PI. Step 2: The transplantation of the PI between rats was established determining the minimal number of PI to reverse the diabetes (glycemia > 300mg/dL) induced by streptozotocin. In addition, the rejection was analyzed by PI survival (time with glycemia <300mg/dL) in syngeneic (Lewis-Lewis) and allogeneic (Sprague-Dawley-Lewis) transplantation. To reverse the diabetes at least 2,500 PI were necessary. Transplantation between syngenic rats (n=6) disclosed no rejection of the PI. In the allogeneic transplantation (n=12), the PI had a short survival (11±1 days). Ten days post-Tx, a higher number of macrophages and T lymphocytes were observed in the grafts, accompanied by very low insulin expression. Step 3: The expression vector for IDO was constructed from RNA extracted from rat placenta. RT-PCR was carried out to amplify the IDO cDNA, which was inserted into expression vector (IDO vector). Step 4: The IDO vector was introduced into PI through lipofection (Lipofectamine 2000) analyzing several concentrations of the IDO vector (0, 0.5, 1.0 and 10 ng/uL) and several periods of incubation (1h, 15h e 24h). The IDO expression in PI was confirmed by RT-PCR and immunohistochemistry. The incubation with 10 ng/uL of IDO vector during 24h was efficient to induce IDO expression in PI. The function of the IDO was confirmed by tryptofan degradation in culture (measurement of tryptofan by HPLC). Step 5: Eleven allogenic transplants (Sprague-Dawley to Lewis) of PI expressing IDO were performed to analyze the effect of the IDO in the rejection. Eight animals were accompanied for 45 days, whereas three were sacrificed after 10 days for immunohistochemistry analysis. Finally, the survival of the PI expressing IDO was significantly higher than nontransfected PI. The study concludes that the induction of the IDO into PI protects the PI increasing the PI survival.

Les douleurs neuropathiques, celles qui sont provoquées par des lésions du système nerveux central ou périphérique, sont les plus difficiles à traiter du fait de leur résistance aux traitements antalgiques classiques. Les traitements utilisés aujourd’hui font appel à des classes thérapeutiques non spécifiquement ciblées sur la douleur, en particulier des antidépresseurs et des anticonvulsivants. Leur efficacité limitée ne repose en fait que sur des observations empiriques. Une meilleure connaissance des processus physiopathologiques sous-tendant les douleurs neuropathiques constitue un préalable à toute innovation thérapeutique, et c’est à cette fin que je me suis appliqué à développer deux modèles de douleurs neuropathiques chez le rat pour en étudier les caractéristiques comportementales, fonctionnelles, cellulaires et biochimiques. Le premier modèle visait à l’induction d’une douleur neuropathique centrale provoquée par la section complète de la moelle épinière au niveau thoracique (T8-T9) ; le second a consisté à injecter, directement au niveau spinal, par voie intrathécale (i.t.), le facteur neurotrophique BDNF (Brain Derived Neurotrophic Factor ; dont l’implication dans les voies de signalisation nociceptive est bien établie dans la littérature). Dans les deux cas, les conséquences pro-algiques de ces interventions ont été comparées à celles induites par la ligature unilatérale du nerf sciatique, qui constitue encore aujourd’hui un modèle classique, mais très imparfait, d’une douleur neuropathique périphérique. Dès le 2ème jour après la section spinale, et jusqu’au moins deux mois plus tard, les rats lésés présentent une forte allodynie mécanique (test des filaments de von Frey) dans le territoire cutané juste en avant de la lésion. Cet effet traduit bien une neuropathie centrale car il n’existe pas chez les rats « sham » qui ont subi l’intégralité de l’intervention chirurgicale à l’exception de la section spinale. L’allodynie mécanique est associée à une induction significative de l’expression (RTqPCR) de marqueurs de souffrance neuronale (ATF-3) et d’activation microgliale (OX-42, récepteurs P2X4, P2X7 et TLR4) et astrocytaire (GFAP), ainsi que du BDNF et de cytokines pro-inflammatoires (IL-1ß, IL-6, TNF-α), mais de façon plus transitoire, ceci dans les ganglions de racines dorsales et/ou la moelle épinière dorsale (comme à la suite de la ligature du nerf sciatique, mais avec des cinétiques différentes). Pour sa part, l’injection intrathécale i.t. d’une dose infra-nanomolaire unique de BDNF (0.3 – 3.0 ng) induit aussi une forte allodynie et une hyperalgésie mécaniques, au niveau des pattes postérieures, qui se développent en 3-5 jours, et perdurent pendant deux semaines. Cependant, au contraire de la section spinale (et de la ligature du nerf sciatique), l’injection i.t. de BDNF ne provoque pas d’activation microgliale ni d’induction de cytokines. Elle entraine en revanche une auto-induction du BDNF, qui semble clé pour l’hyperalgésie puisque celle-ci peut être, en grande partie, supprimée par l’administration d’un inhibiteur du récepteur TrkB du BDNF, la cyclotraxine B (20 mg/kg i.p.), comme d’ailleurs l’hyperalgésie induite par la ligature du nerf sciatique. Au plan pharmacologique, un antalgique opiacé comme le tapentadol s’est révélé efficace dans les deux modèles. De même, les anticonvulsivants, comme la prégabaline et la gabapentine, ont réduit la douleur neuropathique chez les rats injectés par le BDNF i.t. et chez les rats CCI-SN. En conclusion, il semble que l’injection intrathécale de BDNF, qui évite la réalisation de lésions par intervention chirurgicale, puisse constituer un nouveau modèle pertinent de douleur neuropathique chez le rat. De plus, nos résultats laissent à penser que le blocage de la voie de signalisation BDNF-TrkB pourrait ouvrir de nouvelles pistes pour la réduction des douleurs neuropathiques périphériques. (...) / Neuropathic pain, caused by lesions of central or peripheral nervous system, is difficult to treat because of its resistance to classical antalgic treatments. Most of pharmacotherapeutic treatments of neuropathic pain (antidepressants, anticonvulsants) currently used are only based on empirical data and are not specifically aimed at relieving pain. Better knowledge of the mechanisms underlying neuropathic pain is an absolute prerequesite to develop new and innovative treatments. With the aim of contributing to elucidate these mechanisms, I developed two models of neuropathic pain in rats, and studied their behavioral, pharmacological, cellular and biochemical characteristics. The first model consisted of the induction of central neuropathic pain by complete transection of the spinal cord at T8-T9 level. The second one consisted of the administration of BDNF (Brain Derived Neurotrophic Factor; which implication in nociceptive signaling pathways is well established in the literature), directly at the spinal level, via intrathecal (i.t.) injection. In both cases, pro-algesic consequences of these interventions have been compared to those induced by unilateral ligation of the sciatic nerve, which is still considered as a classical, although not really satisfactory, model of peripheral neuropathic pain. From the second day after spinal cord transection up to (at least) 2 months later, lesioned rats developed a strong mechanical allodynia (von Frey filaments test) within a limited cutaneous territory just rostral to the surgical scar. This effect really reflected central neuropathic pain because it did not occur in control, « sham operated » animals, that underwent the same surgical intervention except the spinal cord transection. Mechanical allodynia was associated with marked overexpression of markers of neuronal injury (ATF-3), microglial activation (OX-42, P2X4, P2X7 and TLR4 receptors), astrocyte activation (GFAP), as well as upregulation of transcripts encoding BDNF and pro-inflammatory cytokines (IL-1ß, IL-6 and TNF-α, but only transiently for the latter cytokine), in dorsal root ganglia and/or spinal cord. Therefore, spinal cord transection triggered a strong neuroinflammatory reaction, like that occurring after peripheral nerve lesion, but with different time course and amplitude. On the other hand, intrathecal injection of an infra-nanomolar dose of BDNF (0.3 – 3.0 ng) also induced a strong mechanical allodynia and hyperalgesia at hindpaw level, which developed within 3-5 days and lasted for at least two weeks. However, in sharp contrast with spinal cord transection (and sciatic nerve ligation), i.t. injection of BDNF did not induce any microglial activation and/or proinflammatory cytokines upregulation. Intrathecal (exogenous) BDNF-induced (endogenous) BDNF auto-induction might play a key role in the maintenance of i.t. BDNF-induced hyperalgesia as the latter can be reversed by pharmacological blockade of the BDNF receptor TrkB (with cyclotraxin B at 20 mg/kg i.p., which also reversed sciatic nerve ligation-induced hyperalgesia). Pharmacological investigations showed that the opioid antalgic drug tapentadol and anticonvulsants such as pregabalin and gabapentin efficiently reduced neuropathic pain in i.t. BDNF i.t. as well as in sciatic nerve-ligated rats. Accordingly, intrathecal injection of BDNF might represent a new non-surgical model of neuropathic pain in rats. Moreover, our results indicate that blockade of BDNF-TrkB signaling could open new therapeutic perspectives for alleviating peripheral neuropathic pain. This innovative pharmacological approach should also be explored in the case of central neuropathic pain caused by spinal cord injury.

[pt] Criação seletiva bidirecional de uma resposta defensiva ou qualquer outra característica fenotípica é uma técnica na qual animais são criados com o objetivo de modificar a frequência dos genes que estão subjacentes a um fenótipo em particular. O acasalamento de animais de uma determinada população com base nos extremos opostos de uma característica observável vai propagar, após diversas gerações, este fenótipo particular em direções opostas, levando-se à criação de duas linhagens contrastantes. No presente trabalho empregamos o congelamento condicionado em resposta a estímulos contextuais previamente associados com choques elétricos nas patas como critério de seleção para o desenvolvimento de duas novas linhagens de ratos. O protocolo básico consistiu de acasalamento entre machos e fêmeas Wistar com as maiores e as menores taxas de congelamento condicionado em resposta a sinais contextuais da câmara experimental onde os animais foram expostos a três choques elétricos não sinalizados no dia anterior. O Estudo 1 apresenta os resultados iniciais de quatorze gerações de criação seletiva. Os resultados mostraram que diferenças significativas entre estas duas linhagens foram encontradas após 3 gerações, indicando um forte componente hereditário deste tipo de aprendizagem. As linhagens foram denominadas Cariocas com Alto Congelamento Condicionado (CAC) e Cariocas com Baixo Congelamento condicionado (CBC). Além disso, nós introduzimos um terceiro grupo de animais aleatoriamente selecionados (CTRL) em nosso programa de criação seletiva. No Estudo 2 investigamos os diferentes padrões de extinção e da reaquisição do medo condicionado nestas duas novas linhagens. Por fim, no Estudo 3, nossos resultados sugeriram uma dissociação entre o medo contextual e o medo discreto entre animais CAC e CBC. / [en] Bidirectional selective breeding of a defensive response or any other phenotypic characteristic is a technique in which animals are bred to modify the frequency of the genes that underlie a particular phenotype. Mating animals within a population based on the opposite extremes of an observable characteristic will push, over many generations, this particular phenotype in opposite directions, leading to two separately bred lines. In the present work we employed the conditioned freezing in response to contextual cues previously associated with footshock as the phenotype criterion for developing two new rat lines. The basic protocol consisted of mating male and female albino Wistar rats with the highest and lowest conditioned freezing in response to the contextual cues of the experimental chamber where animals were exposed to three unsignaled electric footshocks on the previous day. Study 1 presents the initial results of fourteen generations of selective breeding. We found that after three generations, reliable differences between these two lines were already present, indicating a strong heritable component of this type of learning. The lines were named Carioca High conditioned Freezing (CHF) and Carioca Low conditioned Freezing (CLF). Also, we introduced a third group of randomly selected animals (RND) in our selective breeding program. In Study 2, we investigated the different patterns of fear extinction and reacquisition in these two new lines. Finally, in Study 3, results showed dissociation between contextual and phasic fear between CHF and CLF rats.

La restriction alimentaire chronique correspond à un des troubles du comportement alimentaire observé en particulier dans l’anorexie mentale (AN) de type restrictif, pathologie qui touche essentiellement les adolescentes et les jeunes femmes. En plus de ce comportement restrictif, une activité physique importante est observée chez un grand nombre de patientes (40 à 80% des cas). Cette maladie se traduit par de nombreuses altérations physiologiques comme des perturbations neuroendocrines, métaboliques, osseuses (ostéopénie, ostéoporose) et ce quelle que soit la cause psychiatrique qui a conduit au développement de ce comportement. De plus, de nombreux arguments suggèrent que l’AN pourrait être considérée comme un trouble « addictif » qui se manifesterait par une addiction à la perte de poids et/ou à la restriction alimentaire ou encore à l’activité physique suggérant une altération du système dopaminergique de récompense. Ainsi, quelles que soient les origines de la maladie, l’AN entraîne des perturbations périphériques et centrales susceptibles d’être impliquées dans une première phase dite « d’adaptation » permettant aux malades de survivre à ces conditions drastiques. Les patients peuvent par la suite tomber dans une seconde phase de « chronicisation » dans laquelle ces mêmes facteurs pourraient être responsables de la dégradation de l’état des malades et conduire, dans les cas les plus graves d’épuisement, à la mort.Notre étude a comme pivot la ghréline, hormone orexigène, dont les concentrations plasmatiques sont augmentées significativement chez les patients anorexiques. Sécrétée principalement en périphérie par les cellules de l’estomac, elle va cibler plusieurs organes aussi bien périphériques que centraux. En particulier, au niveau périphérique, cette hormone agit au niveau du foie dont la principale fonction connue est le maintien de l’homéostasie glucidique. Elle agit également au niveau du tissu adipeux qui est alors stimulé, favorisant ainsi sa croissance avec un stockage des réserves et au niveau musculaire en entrainant entre autre une diminution des réserves de triglycérides. Au niveau du système nerveux central, parmi les sites d’action de la ghréline, on trouve les structures impliquées aussi bien dans le contrôle, qualifié d’homéostatique, de la prise alimentaire représenté par l’hypothalamus, que dans le contrôle, dit hédonique (motivation/récompense), de ce même comportement correspondant au circuit méso-limbique. Pour étudier son implication dans les mécanismes adaptatifs, et éventuellement, dans l’aggravation de la maladie, nous avons mis au point un modèle animal de restriction alimentaire chronique mimant les symptômes physiologiques de l’AN. Notre premier objectif a été de caractériser (« phénotypage ») ce modèle sur le plan physiologique (métabolique, endocrinien) afin de l’utiliser pour notre deuxième objectif : évaluation du rôle de la ghréline comme potentiel facteur prédictif de l’évolution de la maladie.Les données obtenues valident notre modèle comme un modèle pertinent pour étudier sur le long terme les altérations physiologiques et centrales décrites dans l’AN de type restrictif. Nous montrons que l’exercice physique modéré associé à la restriction alimentaire a des effets stabilisateurs sur de nombreux paramètres métaboliques limitant ainsi un épuisement prématuré des ressources énergétiques. En ce qui concerne la ghréline, les concentrations plasmatiques élevées observées dans notre modèle pourraient contribuer également à une régulation adaptative du métabolisme énergétique. / Chronic food restriction is one of the major features observed in anorexia nervosa (AN), especially in the restrictive type. This major eating disorder affects mainly teenager girls and young women. Additionally to the restriction behavior, important physical activity is observed in a large number of patients (40-80% of cases). This disease induces various physiological alterations that concern neuroendocrine, metabolic and bone (osteopenia, osteoporosis) pathways, which have dramatic consequences on the patient’s health. Moreover, many arguments suggest that AN could be considered like an "addictiv" disorder supported by an addiction to weight loss and/or food restriction or physical activity. It thus suggests modifications of the central dopaminergic reward system. Furthermore, whatever the origins or the causes of this disorder, AN leads to peripheral and central alterations that might be involved in an "adaptation" phase allowing patients surviving to these drastic conditions. For some patients, a phase of "chronicity" is described in which these physiological changes may worsen the patient conditions and contribute, when exhaustion is amplifying, to death. Our study points out ghrelin, an orexigenic hormone whose plasma concentrations are significantly increased in AN patients. Mainly secreted by stomach cells, it targets multiple peripheral organs as well as numerous neuronal structures in the brain. At the peripheral level, this hormone acts among others in the liver whose main function is the maintenance of glucose homeostasis. It acts also on the adipose tissue to promote its growth that is associated with lipid storage and on muscle resulting in a reduction of triglycerides stock. At the central nervous system level, ghrelin have various targets like the structures involved in the homeostatic as well as hedonic (motivation/reward) control of food intake through the hypothalamus and the meso-cortico-limbic system respectively.To study the involvement of ghrelin in the potential adaptive mechanisms, and even in the worsening of the disease, we have developed an animal model of chronic food restriction associated or not with physical activity, mimicking the physiological symptoms of AN. Our first objective was to characterize and to phenotype the mouse model by evaluating various physiological (metabolic, endocrine) factors in order to study in our second objective the role of ghrelin as a potential predictor of disease progression.We showed that our mouse model constitutes a pertinent model to study on a long term duration the physiological and central altérations described in the restrictive type AN. Moreover, we showed that moderate physical activity associated with food restriction had stabilizing effects on numerous metabolic parameters that may reduce an early exhaustion of energy stocks. Concerning the role of ghrelin in such model, its plasma concentrations were increased like in AN patients and were suggested to contribute to the adaptive regulation of energy metabolism.