In vitro degradation and in vivo biocompatibility of chitosan-poly(butylene succinate) fiber mesh scaffolds

In tissue engineering, the evaluation of the host response to the biomaterial implantation must be assessed to determine the extent of the inflammatory reaction. We studied the degradation of poly(butylene succinate) and chitosan in vitro using lipase and lysozyme enzymes, respectively. The subcutaneous implantation of the scaffolds was performed to assess tissue response. The type of inflammatory cells present in the surrounding tissue, as well as within the scaffold, was determined histologically and by immunohistochemistry. In the presence of lipase or lysozyme, the water uptake of the scaffolds increased. Based on the weight loss data and scanning electron microscopy analysis, the lysozyme combined with lipase had a notable effect on the in vitro degradation of the scaffolds. The in vivo implantation showed a normal inflammatory response, with presence of neutrophils, in a first stage, and macrophages, lymphocytes, and giant cells in a later stage. Vascularization in the surrounding tissue and within the implant increased with time. Moreover, the collagen deposition increased with time inside the implant. In vivo, the scaffolds maintained the structural integrity. The degradation in vitro was faster and greater compared to that observed in vivo within the same time periods.Ana R Costa-Pinto was supported by the scholarship SFRH/24735/2005 from the Portuguese “Fundação para a Ciência e a Tecnologia” (FCT). This work was partially supported by the European Network of Excellence EXPERTISSUES (NMP3-CT-2004-500283) and FCT funded project Maxbone (PTDC/ SAU-ENB/115179/2009)

“PhenoWorld”: a new multidimensional strategy for studying behaviour in rodents

Tese de Doutoramento em Ciências da SaúdeAnimal models play a central role in biomedical sciences having an important role in the progress of knowledge regarding the pathophysiology of neuropsychiatric disorders, as well as in the development of treatment strategies and testing new drugs for human application. One of the more prevalent neuropsychiatric disorders, causing serious medical and social problems, is depression. The full aetiology of this multidimensional and heterogeneous disorder, as well as its neurobiology, are not yet understood, and the diagnosis of depression in itself is based on multiple symptoms or psychopathological phenomena that together characterize a depressive state. Furthermore, the efficacy of antidepressive treatments is not always similar for different patients and the neurobiological mechanisms of action of commonly used antidepressant drugs are also not yet fully understood. The need to further investigate the psychopathology of the disorder and the mechanisms underlying the actions of antidepressant drugs and treatments, and the recognition that there are many fundamental physiological and behavioural responses that have been evolutionarily conserved between species, depending on similar neural structures and circuits, has resulted in scientists developing and validating several approaches to study depression. These include mimicking the disorder in animal model systems and using several tests to screen for the “symptoms” or depressive-like behaviours. Still, these models and tests present several limitations and their poor predictive power has been argued to be one of the reasons for the lack of efficacy of antidepressant drugs in humans. In face of the drawbacks and limitations presented by test paradigms in use in the field of behavioural neuroscience, and recognizing the need to improve those paradigms for the progress in the knowledge of the pathophysiology and therapeutics of mood disorders, we developed and validated a new paradigm for testing and analysing behavioural data – the “PhenoWorld” (PhW). This new paradigm is a refined housing setup, where groups of 6 rats with implanted RFID transponders live in an ethological enriched setting and have their feeding, locomotor activity, resting and social behavior automatically monitored. In a first study, we assessed the face and predictive validity of our new paradigm using a battery of emotional and cognitive standard tests in order to characterize the behavioral phenotype of animals living in the PhW and in standard conditions (in groups of 6 and 2 rats), after exposure to an unpredictable chronic mild stress (uCMS), and after exposure to uCMS and antidepressant (fluoxetine) treatment. We have shown that animals living in the PhW displayed similar, but more striking, behavioral differences when exposed to uCMS, such as increased behavioral despair shown in the forced swimming test, resting/sleep behaviour disturbances and reduced social interactions, compared to standard housed animals. Moreover, several PhW-cage behaviors, such as spontaneous will to feed or to exercise in running wheels, proved to be sensitive indicators of depressive-like behavior. In a second study, we assessed neuronal plasticity as a consequence of living in the PhW in a group of 6 animals, by comparing the structure of the prefrontal cortex (PFC) and the nucleus accumbens (NAc), two brain areas involved in the circuitry regulating motivation and reward, of PhW animals compared to those housed in standard conditions in social groups of 6 (STD6) and 2 rats (STD2). Findings indicated that rats living in the PhW displayed increased medial PFC layer II volumes, increased branching and length, as well as increased spine densities and spine numbers per neuron, in medial PFC pyramidal neurons. In the NAc, significant increases were only found for the length of NAc shell medium spiny neurons. The results proved the construct validity of the PhW and revealed that living in a naturalistic complex environment, closer to ‘real life experience’, impacts on the structure of brain regions implicated in complex multidimensional disorders. Finally, we addressed the animal welfare comparing the three housing conditions: PhW, STD6 and STD2. In this study, home-cage behaviour analysis showed that PhW animals have a clearer circadian pattern of sleep and social interactions. By promoting a good basic health and functioning, and the performance of natural behaviours with controllability but still keeping some physical and social challenges, the PhW stimulates positive affective states and higher motivation in rats. Even though understanding animal welfare has always some degree of subjectivity, these are evidences pointing for an increased welfare of animals living in the PhW. In conclusion, after validating the PhW to study a multidimensional mood disorder, we believe this new paradigm and naturalistic housing setting allows to address real systems neuroscience and can be very useful in future animal studies of other complex emotional and cognitive disorders.Os modelos animais têm um papel preponderante nas ciências biomédicas contribuindo para o progresso no conhecimento da patofisiologia das doenças neuropsiquiátricas e para o desenvolvimento de estratégias de tratamento, bem como para testar novos medicamentos em ensaios pré-clínicos. Uma das doenças psiquiátricas mais prevalentes, caracteristicamente multidimensional e heterogénea e que causa graves problemas médicos e sociais, é a depressão. Nem a etiologia, nem a neurobiologia desta doença são ainda completamente conhecidas e o diagnóstico de depressão é feito com base em múltiplos sintomas ou fenómenos psicopatológicos que no seu conjunto caracterizam o estado depressivo. A eficácia dos tratamentos antidepressivos não é semelhante para todos os pacientes, e os mecanismos neurobiológicas de ação dos tratamentos e fármacos antidepressivos não são ainda totalmente conhecidos. A necessidade de continuar a investigar a psicopatologia da depressão e os mecanismos pelos quais os antidepressivos exercem os seus efeitos, bem como o reconhecimento de que existem muitas respostas fisiológicas e comportamentais evolucionariamente conservadas entre espécies, levou os cientistas a desenvolver e validar estratégias de estudo da depressão mimetizando a doença em modelos animais e usando diversos testes para avaliar comportamentos tipo depressivos ou “sintomas”. No entanto, os testes animais desenvolvidos apresentam diversas limitações e um poder preditivo baixo, contribuindo para a baixa eficácia dos fármacos antidepressivos em humanos. Tendo em conta todas as limitações apresentadas e reconhecendo a necessidade de melhorar os paradigmas de comportamento animal usados em neurociências no sentido de promover o progresso no conhecimento da patofisiologia e terapêutica das doenças emocionais, desenvolvemos e validamos um novo paradigma para testar e analisar dados comportamentais de ratos – o “PhenpoWorld” (PhW). Este novo paradigma providencia um sistema de alojamento melhorado onde grupos de 6 ratos, com um microship emissor de radiofrequência implantado subcutaneamente, vivem num ambiente etologicamente mais rico e onde têm os seus comportamentos alimentares, atividade locomotora, e comportamento social automaticamente monitorizado. No primeiro estudo realizado, avaliamos as validades aparente e preditiva do novo paradigma utilizando uma bateria de testes de comportamento emocional e cognitivo de forma a caracterizar o fenótipo dos animais a viver no PhW ou em gaiolas convencionais (em grupos de 6 ou de 2 ratos), após exposição crónica a stress ( uCMS) ou após exposição a uCMS e tratamento com fluoxetina. Os resultados obtidos mostraram que os animais que vivem no PhW apresentam diferenças comportamentais semelhantes, mas mais vincadas, quando expostos a stress, tais como distúrbios do padrão de sono/descanso e redução da frequência de interações sociais. Além disso, verificamos que vários comportamentos apenas possíveis no PhW (vontade espontânea de atravessar portas automáticas para ir comer e ir fazer exercício nas rodas) são indicadores sensíveis de comportamento tipo depressivo. No segundo estudo, avaliamos a plasticidade neuronal como consequência de viver no PhW num grupo de 6 animais, comparando a estrutura do córtex pré-frontal ( PFC) e do nuccleus accumbens ( NAc), duas áreas do cérebro envolvidas nos circuitos que regulam motivação e recompensa, entre esses animais e animais alojados em gaiolas convencionais em grupos de 6 (STD6) e de 2 ratos (STD2). Os resultados revelaram, por exemplo, que os ratos que vivem no PhW apresentam um aumento do volume da camada II do PFC medial, e um aumento da complexidade das dendrites e do nº de espinhas por neurónio nos neurónios piramidais da camada II-III do PFC medial. No NAc, apenas verificamos aumentos significativos no comprimento das dendrites dos neurónios espinhosos médios da subdivisão NAc shell. Estes resultados refletem a validade construtiva do PhW e revelam que viver nesse ambiente naturalístico complexo, mais parecido com a vida real, tem impacto nas áreas do cérebro implicadas nas doenças multidimensionais complexas. Finalmente, avaliamos o bem-estar animal comparando as três condições de alojamento (PhW, STD6 e STD2) e mostramos que os padrões circadianos de sono e das interações sociais são mais marcados no PhW. A promoção da saúde, e o estímulo de comportamentos naturais com controlabilidade mas mantendo alguns desafios físicos e sociais contribuem para um estado afetivo positivo dos animais no PhW e para a sua elevada motivação e são indicadores de elevado bem-estar animal. Em conclusão, após validação do PhW no estudo de um modelo de depressão, acreditamos que este novo paradigma e gaiola naturalista permita futuros estudos no âmbito das neurociências de sistemas tornando-se muito útil em estudos animais de outras doenças emocionais e cognitivas complexas

The impact of light, noise, cage cleaning and in-house transport on welfare and stress of laboratory rats

Human interaction and physical environmental factors are part of the stimuli presented to laboratory animals everyday, influencing their behaviour and physiology and contributing to their welfare. Certain environmental conditions and routine procedures in the animal facility might induce stress responses and when the animal is unable to maintain its homeostasis in the presence of a particular stressor, the animal's wellbeing is threatened. This review article summarizes several published studies on the impact of environmental factors such as light, noise, cage cleaning and in-house transport on welfare and stress of laboratory rats. The behaviour and physiological responses of laboratory rats to different environmental housing conditions and routine procedures are reviewed. Recommendations on the welfare of laboratory rats and refinements in experimental design are discussed and how these can influence and improve the quality of scientific data