Células-tronco em modelo de lesão cutânea infectada : efeito antimicrobiano e alterações na cicatrização

Introdução: Células-tronco mesenquimais podem atuar como facilitadores dos processos de reparação e regeneração suprimindo a liberação de citocinas pró-inflamatórias, estimulando aquelas de natureza anti-inflamatória e atuando na regulação da resposta a infecções. Apesar destes efeitos estarem insuficientemente caracterizados, sabe-se que o reconhecimento de bactérias por receptores específicos da membrana celular desencadeia uma resposta inflamatória e imune no tecido que tem o potencial de modular o processo de reparação, tornando essas células atraentes candidatas em aplicações terapêuticas. Objetivos: Este estudo avaliou a aplicação de células-tronco mesenquimais, derivadas de tecido adiposo de suíno, em feridas experimentalmente induzidas e contaminadas, quanto à sua capacidade antimicrobiana e alterações no processo de cicatrização cutânea in vivo. Também avaliou o metabolismo, proliferação celular e secreção de peptídeo antimicrobiano PMAP-23 em células tronco adiposo derivadas de suínos in vitro, mediante estímulo com Lipopolisacarídeo (LPS). Métodos: Para os ensaios in vivo 8 suínos foram divididos em dois grupos: Controle (C) (solução salina) ou ADSC (células tronco). Foram confeccionadas seis feridas de espessura total com 4 cm de diâmetro na região dorsal dos animais, infectadas experimentalmente com E.coli (108 CFU em 100μL de solução salina). Nos dias 3,5, e 7 pós infecção as lesões do grupo ADSC foram tratadas com 106 cels/mL (P3) em solução salina. Foram realizados testes histopatológicos, imunohistoquimicos e de força tensil nas feridas, para mensurar parâmetros de qualidade cicatricial. Para a realização dos ensaios in vitro as culturas celulares (1×104 cels/cm2 em P3) foram estimuladas com adição de 10 μg/mL de LPS de E. coli 0111:B4 em meio de cultura completo e incubadas por 1 ou 7 dias. As células do grupo controle foram suplementadas com PBS para controle do veículo e mantidas nas mesmas condições. Para a determinação de proliferação celular foram submetidas à ensaio de SRB, para a a atividade metabólica foi realizado ensaio de MTT e a quantificação de PMAP-23 foi mensurada por ELISA. Resultados: Foi possível verificar, nos experimentos in vivo que a força tensil das cicatrizes do grupo tratado foi significativamente maior (p<0,001) aos 21 dias de análise. Também houve aumento significativo (p=0.03) na concetração de PMAP-23 no D3 do grupo tratado com ADSC. Nas análises de resposta inflamatória, taxa de contração e tempo de fechamento da ferida não houve diferença significativa entre os grupos. Nos experimentos in vitro observamos aumento significativo (p=0.001) na atividade mitocondrial mediante exposição prolongada ao LPS e aumento significativo da secreção de PMAP-23 (p<0.04). Conclusões: Os resultados encontrados sugerem que as toxinas produzidas pelas bactérias não impactam negativamente nas propriedades de imunomodulação e cicatrização das células mesenquimais, possibilitando a aplicação de terapia celular em ambientes de ferida contaminada.Background: Adipose-derived mesenchymal stem cells (ADSCs) are characterized by their modulation of host-immune responses ability. On the basis of these properties, ADSCs are currently under clinical investigation in a range of therapeutic applications. More recently, its antibacterial activity has also been demonstrated. However, data on the direct interaction between MSCs and microbial pathogens are sparse, it has been demonstrated that MSCs exhibit a cell autonomous, broad-spectrum antimicrobial effector function. Objectives: We evaluate the effect of Escherichia coli infection on the biomechanical strength and histological quality in a porcine in vivo model of wound healing and investigated the impact of bacterial lipopolysaccharide (LPS) on proliferation, metabolic activity and PMAP-23 functional assays of mesenchymal stem cell cultures derived from porcine adipose tissue. Methods: For in vitro assays eight animals was randomly divided in two groups: Control and ADSC. Under general anesthesia, a total of six 4cm (diameter) full-thickness cutaneous lesions were surgically inflicted on dorsal surfaces and at days 3, 5 and 7 each lesion was intradermally injected with either saline or ADSC (106 cells/mL). Biopsies were assessed for histopathological, immunohistochemical and tensile strength analysis. For in vitro analisys ADSCs, in P3, (1×104 cells/cm2) were incubated with 10 μg/mL lipopolysaccharide (LPS) from E coli 0111:B4 for the LPS group. Incubation with bacterial endotoxin was maintained for short-term (1-day post-challenge: D1) or long-term (7-days post-challenge: D7). After the exposure period to bacterial toxins, cells were tested to determinate proliferation (SRB assay), metabolic activity (MTT assay) and PMAP-23 quantification (ELISA). Results: The tensile strength of the wound showed significant improvement (p<0,001) at 21 days of healing. There were significant differences (p=0.03) between groups in PMAP-23 concentration at D3, with increased concentration levels found in ADSC – treated group. There was no significant difference in wound contraction percentages, time of healing and inflammatory response for CTRL and ADSC group. In in vitro assays cell proliferation was not affected by LPS, while mitochondrial metabolic activity was promoted p=0.001) by prolonged exposure to LPS. In addition, exposure to LPS induced a significant higher PMAP-23 p<0.04) secretion. Conclusions: collectively, these findings suggest that toxins produced by bacteria do not affect the intrinsic properties of ADSCs and consequently, application of stem-cell therapies in the context of infection may have therapeutic effect

Adjuvants for skin healing

Os adjuvantes da cicatrização são alternativas utilizadas pelos cirurgiões e/ou clínicos para ajudar na reparação dos tecidos. As tentativas humanas de intervir no processo da cura das feridas remontam à antiguidade. A cicatrização de feridas é um evento complexo que ocorre espontaneamente, mas quando tratada tende a ocorrer de forma mais rápida e com melhores resultados funcionais e estéticos. A necessidade ou escolha de um tratamento para a ferida depende da etiopatogenia, dos fatores locais e sistêmicos. Por esses motivos, a intervenção de qualquer ferida deve ser individualizada e o produto de escolha do curativo deve ser avaliado cuidadosamente, correlacionando indicações, contraindicações, eficácia, custo e benefício.Processing aids healing alternatives are used by surgeons and/or clinical trials to aid in tissue repair. The human attempts to intervene in the process of healing the wounds date back to antiquity. Wound healing is a complex event that occurs spontaneously, but when treated tends to occur faster and with better functional and aesthetic results. The necessity or choice of treatment for a wound depends on the etiology, the local and systemic factors. For these reasons, the intervention of any wound should be individualized and the product of choice of dressing should be evaluated carefully, correlating indications, contraindications, efficacy, cost and benefit

Feline injection-site sarcoma

O sarcoma de aplicação felino se caracteriza como uma neoplasia maligna de fibroblastos e é atualmente um grande desafio para o médico veterinário. Aplicações de vacinas e medicações injetáveis por via subcutânea ou intramuscular aparecem como iniciadoras do processo dessa neoplasia, mais precisamente a inflamação persistente causada pelo fármaco ou antígeno administrado. Seu diagnóstico baseia-se nos sinais clínicos, avaliação citológica e principalmente histopatológica. O tratamento mais eficaz ainda não está estabelecido, mas acredita-se que a multimodalidade de terapias como: cirurgia, radioterapia e quimioterapia sejam as opções mais indicadas. O conhecimento da afecção em todos os seus aspectos irá fornecer aos colegas profissionais subsídios em relação a melhor maneira de abordá-la em termos de diagnóstico, tratamento e prevenção.The feline injection-site sarcoma characterize as malignant neoplasia of fibroblastos and is a challenge for the veterinarian. The injectable applications (vaccines, medications) seems to be the reason for that neoplasia, more specifically, the inflammation caused by injury of given drugs or antigens. Its diagnosis is based on the clinical signals, cytological and mainly histopathology evaluation. The most effective treatment has not been established yet, but it is believed that a multimodality of therapies how: surgery, radiotherapy and chemotherapy would be the most indicated options. The knowledge of the illness in all of its aspects will supply to professionals colleges subsidies in relation to the best way to approach its diagnosis and treatment

Arachnid accident in dog : case report

Os acidentes causados por aranhas são frequentes em nosso meio. A Phoneutria sp. é conhecida popularmente por aranha-armadeira e considerada a mais perigosa do Brasil. Estas aranhas são responsabilizadas pelo maior número de acidentes aracnídeos notificados ao Ministério da Saúde. Foi atendido no Hospital de Clínicas Veterinária da Universidade Federal do Rio Grande do Sul (HCV-UFRGS) um canino, cocker spaniel, 11 meses de idade, picado por aranha do gênero Phoneutria sp., que foi internado e submetido às terapias necessárias. O animal apresentando sinais clínicos de acidente aracnídico de grau grave, pela indisponibilidade, não recebeu o soro antiaracnídico, indo a óbito por choque neurogênico.Accidents caused by spiders are very frequent in our practice. The Phoneutria sp is popularly known by “aranha armadeira” and is considered the most dangerous of Brazil. These spiders are responsible by the biggest number of arachnids accidents notified to the Health Ministry. A canine, cocker spaniel, 11-months-old, pricked by Phoneutria sp. spider was referred to the Hospital de Clínicas Veterinárias, Universidade Federal do Rio Grande do Sul (HCV-UFRGS), addmitted in and medicated. The animal presented signals of arachnid accident of serious degree, and for not having the antiarachnid serum, it came to death by neurogenic shock

Liquid and Gel Platelet Rich Plasma as Skin Healing Adjuvant

Background: In recent decades, many researches have been conducted on processes involved in tissue repairing, mainly in the development of resources and technology designed to improve the wound healing progress. Platelet rich plasma (PRP) derived from autologous blood is defined as a plasma volume with platelet concentration higher than physiological level. It is an autogenous and low cost source of growth factors, which are essential for tissue regeneration due to their angiogenic, mitogenic, and chemotactic properties. The aim of this study was evaluate two forms of PRP- liquid and gel - regarding their capacity to influence quality and repair time of standardized skin injuries.Materials, Methods &amp; Results: New Zealand healthy rabbits were distributed in three groups (n = 6): control group (CG), liquid platelet rich plasma group (LIQPRP), and gel platelet rich plasma group (GELPRP). Acute skin lesions were inducted in two areas approximately 2 cm close to scapular edge and depth including epidermis, dermis, and hypodermis to external muscular fascia. Animals received treatment according to each group. Injuries were measured with digital pachymeter in two directions: longer length (l) and longer width (w), every two days. Areas and healing rates were calculated. Microscopic analysis samples were collected on days seven and 14 and evaluated through hematoxylin and eosin staining (HE) for global tissue examination, and through Masson’s trichrome (MT) to collagen fibers present within the interstice. These analyzes considered: angiogenesis, inflammation infiltrated and collagen fibers quantity. Immunohistochemistry with anti-Ki-67 antibody was utilized for proliferative profile assessment. Kruskal-Wallis’ non-parametric tests of independent samples was performed for comparison of values obtained through platelet count, referring to evaluation of platelet increase on treatments. Scar contraction rate (CR) was evaluated through Shapiro-Wilk’s normality test, and then submitted to mixed models test. Results obtained by histopathological and immunohistochemistry were also evaluated by Shapiro-Wilk’s normality test (for all tests a 5% level of significance was considered). Platelet concentration achieved with liquid PRP was 8.64 and gel PRP reached 5.62 times higher than physiological values. Platelet increase mean for both groups was 7.95. No statistical significance was observed between groups. No side-effects or adverse reactions related to PRP usage were observed while study was conducted.Discussion: In the present study, there was a need to raise platelet poor plasma volume in order to obtain autogenous thrombin required for gel PRP. After this modification, a stable and reasonable platelet concentration gel was produced. However, this form of PRP application requires more time for sample preparation, increasing the production cost. Furthermore, injection of liquid PRP directly in the wound site activates platelets by generated substances due to needle perforation, and mainly due to tissue trauma generated at the lesion site. Relating to the therapies administered, gel PRP was considered more manageable, since 3D structure could easily adapt to wound site after simply deposition of it. Liquid PRP was administered with needle and syringe, which required the surgeon to be more careful and perform a slow injection in order to avoid any spill and loss of material. Furthermore, histopathological analysis did not point any clot traces formed by gel PRP dehydration, although it is not possible to ensure that the clot was eliminated, reabsorbed, or even removed by the animal. By this protocol, a stable and reasonable platelet concentration gel was produced. Further studies are encouraged as well as employment of alternative diagnostic tools, in order to better understand found results

Development of different degrees of elastase-induced emphysema in mice: a randomized controlled experimental study

Introduction: Mouse models of emphysema are important tools for testing different therapeutic strategies. The aim of this study was to develop a mouse model of emphysema induced by different doses of elastase in order to produce different degrees of severity.Methods: Thirty female mice (C57BL/6) were used in this study. Different doses of porcine pancreatic elastase were administered intratracheally once a week for four weeks, as follows: 0.1 U (n=8), 0.15 U (n=7), and 0.2 U (n=7). Control mice (n=8) received 50 microL of sterile saline solution intratracheally. Lung mechanics were analyzed by plethysmography. Mean linear intercept and volume fraction occupied by collagen and elastic fibers were determined.Results: An increase in lung resistance was observed with 0.2 U of elastase [median (P-25-P75): 2.02 (1.67; 2.34) cmH2O.s/mL], as well as a decrease in tidal volume and minute ventilation. Peak expiratory flow increased significantly in the groups treated with 0.15 U and 0.2 U of elastase. Mean linear intercept was higher with 0.15 U and 0.2 U of elastase, with destruction of alveolar walls [median (P-25-P75): 30.31 (26.65-43.13) microm and 49.49 (31.67-57.71) microm respectively]. The volume fraction occupied by collagen and elastic fibers was lower in the group receiving 0.2 U of elastase.Conclusion: Four intratracheal instillations of 0.2 U of elastase once a week induced changes in lung function and histology, producing an experimental model of severe pulmonary emphysema, whereas 0.15 U resulted in only histological changes.Introduction: Mouse models of emphysema are important tools for testing different therapeutic strategies. The aim of this study was to develop a mouse model of emphysema induced by different doses of elastase in order to produce different degrees of severity. Methods: Thirty female mice (C57BL/6) were used in this study. Different doses of porcine pancreatic elastase were administered intratracheally once a week for four weeks, as follows: 0.1 U (n=8), 0.15 U (n=7), and 0.2 U (n=7). Control mice (n=8) received 50 microL of sterile saline solution intratracheally. Lung mechanics were analyzed by plethysmography. Mean linear intercept and volume fraction occupied by collagen and elastic fibers were determined. Results: An increase in lung resistance was observed with 0.2 U of elastase [median (P-25-P75): 2.02 (1.67; 2.34) cmH2O.s/mL], as well as a decrease in tidal volume and minute ventilation. Peak expiratory flow increased significantly in the groups treated with 0.15 U and 0.2 U of elastase. Mean linear intercept was higher with 0.15 U and 0.2 U of elastase, with destruction of alveolar walls [median (P-25-P75): 30.31 (26.65-43.13) microm and 49.49 (31.67-57.71) microm respectively]. The volume fraction occupied by collagen and elastic fibers was lower in the group receiving 0.2 U of elastase. Conclusion: Four intratracheal instillations of 0.2 U of elastase once a week induced changes in lung function and histology, producing an experimental model of severe pulmonary emphysema, whereas 0.15 U resulted in only histological changes

Development of different degrees of elastaseinduced emphysema in mice : a randomized controlled experimental study

Introduction: Mouse models of emphysema are important tools for testing different therapeutic strategies. The aim of this study was to develop a mouse model of emphysema induced by different doses of elastase in order to produce different degrees of severity. Methods: Thirty female mice (C57BL/6) were used in this study. Different doses of porcine pancreatic elastase were administered intratracheally once a week for four weeks, as follows: 0.1 U (n=8), 0.15 U (n=7), and 0.2 U (n=7). Control mice (n=8) received 50 microL of sterile saline solution intratracheally. Lung mechanics were analyzed by plethysmography. Mean linear intercept and volume fraction occupied by collagen and elastic fibers were determined. Results: An increase in lung resistance was observed with 0.2 U of elastase [median (P-25-P75): 2.02 (1.67; 2.34) cmH2O.s/mL], as well as a decrease in tidal volume and minute ventilation. Peak expiratory flow increased significantly in the groups treated with 0.15 U and 0.2 U of elastase. Mean linear intercept was higher with 0.15 U and 0.2 U of elastase, with destruction of alveolar walls [median (P-25-P75): 30.31 (26.65-43.13) microm and 49.49 (31.67-57.71) microm respectively]. The volume fraction occupied by collagen and elastic fibers was lower in the group receiving 0.2 U of elastase. Conclusion: Four intratracheal instillations of 0.2 U of elastase once a week induced changes in lung function and histology, producing an experimental model of severe pulmonary emphysema, whereas 0.15 U resulted in only histological changes

Condrossarcoma mixóide em um coelho

Tumores ósseos primários em animais, com exceção dos osteossarcomas, são raros. O condrossarcoma é uma neoplasia maligna, de crescimento lento, proveniente de tecido cartilaginoso. É um tumor localmente invasivo, tendo grande propensão a recidivas, mas baixa incidência de formação de metástases. O diagnóstico definitivo é dado pelo exame histopatológico. A remoção cirúrgica, com obediência aos preceitos da cirurgia oncológica é o tratamento indicado. Foi atendido, no Hospital de Clínicas Veterinárias da Universidade Federal do Rio Grande do Sul, um coelho, sem raça definida (SRD), macho, não castrado, com aproximadamente três anos de idade e 4,3kg de peso, apresentando claudicação e aumento de volume na região distal do membro pélvico direito. O exame histopatológico revelou diagnóstico de condrossarcoma mixóide de grau II. Realizou-se a amputação do membro afetado. O paciente demonstrou uma boa recuperação dos movimentos nos primeiros dias após a cirurgia, retornando às funções que realizava anteriormente, como caminhar, correr e saltar