Comparison of inbred mouse strains shows diverse phenotypic outcomes of intervertebral disc aging

Intervertebral disc degeneration presents a wide spectrum of clinically degenerative disc phenotypes; however, the contribution of genetic background to the degenerative outcomes has not been established. We characterized the spinal phenotype of 3 mouse strains with varying cartilage-regenerative potential at 6 and 23 months: C57BL/6, LG/J and SM/J. All strains showed different aging phenotypes. Importantly, LG/J mice showed an increased prevalence of dystrophic disc calcification in caudal discs with aging. Quantitative-histological analyses of LG/J and SM/J caudal discs evidenced accelerated degeneration compared to BL6, with cellular disorganization and cell loss together with fibrosis of the NP, respectively. Along with the higher grades of disc degeneration, SM/J, at 6M, also differed the most in terms of NP gene expression compared to other strains. Moreover, although we found common DEGs between BL6 and LG/J aging, most of them were divergent between the strains. Noteworthy, the common DEGs altered in both LG/J and BL6 aging were associated with inflammatory processes, response to stress, cell differentiation, cell metabolism and cell division. Results suggested that disc calcification in LG/J resulted from a dystrophic calcification process likely aggravated by cell death, matrix remodelling, changes in calcium/phosphate homeostasis and cell transformation. Lastly, we report 7 distinct phenotypes of human disc degeneration based on transcriptomic profiles, that presented similar pathways and DEGs found in aging mouse strains. Together, our results suggest that disc aging and degeneration depends on the genetic background and involves changes in various molecular pathways, which might help to explain the diverse phenotypes seen during disc disease. © 2020 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd

A New Perspective on Intervertebral Disc Calcification-From Bench to Bedside

Disc degeneration primarily contributes to chronic low back and neck pain. Consequently, there is an urgent need to understand the spectrum of disc degeneration phenotypes such as fibrosis, ectopic calcification, herniation, or mixed phenotypes. Amongst these phenotypes, disc calcification is the least studied. Ectopic calcification, by definition, is the pathological mineralization of soft tissues, widely studied in the context of conditions that afflict vasculature, skin, and cartilage. Clinically, disc calcification is associated with poor surgical outcomes and back pain refractory to conservative treatment. It is frequently seen as a consequence of disc aging and progressive degeneration but exhibits unique molecular and morphological characteristics: hypertrophic chondrocyte-like cell differentiation; TNAP, ENPP1, and ANK upregulation; cell death; altered Pi and PPi homeostasis; and local inflammation. Recent studies in mouse models have provided a better understanding of the mechanisms underlying this phenotype. It is essential to recognize that the presentation and nature of mineralization differ between AF, NP, and EP compartments. Moreover, the combination of anatomic location, genetics, and environmental stressors, such as aging or trauma, govern the predisposition to calcification. Lastly, the systemic regulation of calcium and Pi metabolism is less important than the local activity of PPi modulated by the ANK-ENPP1 axis, along with disc cell death and differentiation status. While there is limited understanding of this phenotype, understanding the molecular pathways governing local intervertebral disc calcification may lead to developing disease-modifying drugs and better clinical management of degeneration-related pathologies

Suporte de Leitura: desenvolvimento de um protótipo para auxílio a pessoas com baixa visão – testes preliminares

A baixa visão é uma condição intermediária entre a visão normal e a cegueira, secundária a um acometimento irreversível do sistema visual, acarretando prejuízos no desempenho e na realização de determinadas atividades. Para minimizar esses problemas, é recomendada a utilização dos recursos de Tecnologia Assistiva. O objetivo deste trabalho foi fazer um relato do desenvolvimento de um protótipo de Suporte de Leitura (SL) e descrever os resultados dos testes preliminares. Quanto ao aspecto metodológico, foi feito um levantamento dos diferentes dispositivos de auxílio para indivíduos com baixa visão disponíveis no mercado e definiu-se pela construção do protótipo do Suporte de Leitura, que contemplasse as necessidades ergonômicas do usuário, com baixo custo, de fácil acesso, dentro do conceito de design universal. Os resultados obtidos foram: suporte de leitura composto por uma base de sustentação, um tampo reclinável facilitando a portabilidade e trilhos para movimentação do suporte da lente. Concluiu-se que o equipamento desenvolvido possui aspectos positivos, como melhor manutenção da postura, boa usabilidade, portabilidade considerável e boa viabilidade ergonômica. Contudo será fundamental dar continuidade aos testes do equipamento com a população-alvo para garantir a usabilidade e aplicabilidade reais do dispositivo.Low vision is an intermediate condition between normal vision and blindness, secondary to an irreversible impairment of the visual system, causing losses in performance and achievement of certain activities. To minimize these problems, it is recommended the use of Assistive Technology resources. The objective of this paper was to report the development of a Reading Support (RS) prototype and describe the results of the preliminary tests. For the methodological aspect, a survey of different assistive devices available for individuals with low vision was conducted and it was decided to build a Reading Support prototype that considers the ergonomic needs of the user, with low cost, easy access and within the concept of universal design. The results were: reading support comprised of a support base, a reclining top facilitating the portability and rails to drive the lens holder. It was concluded that the equipment developed has positive aspects such as better maintenance of posture, good usability, considerable portability and good ergonomic viability, but it will be essential to continue the equipment testing with the target population to ensure the actual usability and applicability of the device

Development of a standardized histopathology scoring system using machine learning algorithms for intervertebral disc degeneration in the mouse model—An ORS spine section initiative

Mice have been increasingly used as preclinical model to elucidate mechanisms and test therapeutics for treating intervertebral disc degeneration (IDD). Several intervertebral disc (IVD) histological scoring systems have been proposed, but none exists that reliably quantitate mouse disc pathologies. Here, we report a new robust quantitative mouse IVD histopathological scoring system developed by building consensus from the spine community analyses of previous scoring systems and features noted on different mouse models of IDD. The new scoring system analyzes 14 key histopathological features from nucleus pulposus (NP), annulus fibrosus (AF), endplate (EP), and AF/NP/EP interface regions. Each feature is categorized and scored; hence, the weight for quantifying the disc histopathology is equally distributed and not driven by only a few features. We tested the new histopathological scoring criteria using images of lumbar and coccygeal discs from different IDD models of both sexes, including genetic, needle-punctured, static compressive models, and natural aging mice spanning neonatal to old age stages. Moreover, disc sections from common histological preparation techniques and stains including H&E, SafraninO/Fast green, and FAST were analyzed to enable better cross-study comparisons. Fleiss\u27s multi-rater agreement test shows significant agreement by both experienced and novice multiple raters for all 14 features on several mouse models and sections prepared using various histological techniques. The sensitivity and specificity of the new scoring system was validated using artificial intelligence and supervised and unsupervised machine learning algorithms, including artificial neural networks, k-means clustering, and principal component analysis. Finally, we applied the new scoring system on established disc degeneration models and demonstrated high sensitivity and specificity of histopathological scoring changes. Overall, the new histopathological scoring system offers the ability to quantify histological changes in mouse models of disc degeneration and regeneration with high sensitivity and specificity

Lactate efflux from intervertebral disc cells is required for maintenance of spine health.

Maintenance of glycolytic metabolism is postulated to be required for health of the spinal column. In the hypoxic tissues of the intervertebral disc and glycolytic cells of vertebral bone, glucose is metabolized into pyruvate for ATP generation and reduced to lactate to sustain redox balance. The rise in intracellular H+ /lactate concentrations are balanced by plasma-membrane monocarboxylate transporters (MCTs). Using MCT4 null mice and human tissue samples, complimented with genetic and metabolic approaches, we determine that H+ /lactate efflux is critical for maintenance of disc and vertebral bone health. Mechanistically, MCT4 maintains glycolytic and TCA cycle flux and intracellular pH homeostasis in the nucleus pulposus compartment of the disc, where HIF-1α directly activates an intronic enhancer in SLC16A3. Ultimately, our results provide support for research into lactate as a diagnostic biomarker for chronic, painful disc degeneration

Violência doméstica : análise jurídica e perspetiva político-criminal

Exame público realizado em 13 Julho 2015.Dissertação de mestrado realizada no âmbito do Mestrado em Direito.A violência doméstica é um fenómeno complexo que não se esgota na identificação de algumas variáveis mais prováveis para a sua ocorrência. Embora seja um fenómeno cuja visibilidade é maior entre as famílias com fracos recursos económicos e culturais, é extensivo a todas as classes sociais. A questão que queremos analisar consiste em saber se o direito pode e deve ser um instrumento de promoção da igualdade e defesa dos direitos das mulheres e homens que são vítimas deste tipo de violência. Importa saber qual o papel do direito e da lei ao longo do tempo para que, com os exemplos do passado, se possa construir um futuro mais risonho e mais livre para milhares de cidadãos. No nosso trabalho procuramos dar conta da sensibilização para o grave problema que é o stalking, fenómeno que começa a despertar cada vez mais atenções; analisamos, também, as penas acessórias como a cada vez mais utilizada vigilância eletrónica; e centramos a nossa atenção no Instituto da Suspensão Provisória do Processo, a janela que se abriu depois se te ter fechado a porta ao tornar público o crime de violência doméstica. Por último, apresentamos as medidas constantes do V Plano Nacional de Prevenção e Combate à Violência Doméstica e de Género 2014-2017.Abstract: Domestic violence is a complex phenomenon that doesn't end on the identification of few plausible variables. However is it more visible among families with low economical and cultural resources, it is extensible to all social classes. The question we want to analyse consists on foreseeing if the law can and may be an instrument to promote equality on the defence of women and men victims of this kind of violence. It is so important to know the role of law and of the laws throughout time, taking past cases as an example, in order to built a brighter future for thousands of citizens. Our research aims to create awareness for “stalking”, a phenomenon that now starts arising as it also brings up more and more attention. We also intend to analyse the accessory penalties as the electronic monitoring, as one of the most commonly used; we focus our attention on the institute of the Provisory Suspension of the Process, the window opened after closing the door when domestic violence became a public-order crime. Finally, we present the measures included on the 5th National Plan of Prevention and Fight against Domestic and Gender Violence 2014-2017

Contribuição do envelhecimento e da senescência para a degeneração do disco intervertebral

Tese de Doutoramento em Ciências da SaúdeO disco intervertebral é a unidade responsável por absorver o stress mecânico e proporcionar flexibilidade à coluna. Para isso, a composição e organização da matriz extracelular torna.se essencial para preservar o equilíbrio biomecânico deste. Contudo, esta patologia não é devidamente compreendida dada a sua complexidade e falta de modelos experimentais. A degeneração do disco compreende uma vasta apresentação clínica durante o envelhecimento tais como: fibrose, calcificação, hérnia ou apenas degeneração a nível molecular. Para além disto, a relação entre o fundo genético, o envelhecimento e o fenótipo patológico apresentado ainda não foram estabelecidos. Assim, caracterizamos o fenótipo do disco intervertebral de 3 raças de ratinhos com diferentes capacidades regenerativas na cartilagem aos 6 e 23 meses de idade: C57BL/6 (BL6), LG/J e SM/J. Cada espécie de ratinho apresentou uma progressão única de degeneração do disco a nível morfológico e do transcriptoma. Os LG/J mostraram uma grande prevalência de calcificação no disco intervertebral. Por outro lado, os SM/J apresentaram uma degeneração acelerada do compartimento, com desorganização da matriz, morte celular e aumento da deposição de tecido fibrótico em comparação com os BL6. Estes resultados, mostram que a composição genética é um fator essencial no desenvolvimento degeneração do disco e do seu fenótipo durante o envelhecimento. Estudos recentes mostraram também uma associação importante senescência, degeneração do disco e catabolismo de matiz celular, quer em modelos animais quer em humanos. De facto, a eliminação de células positivas para o marcador de senescência, p16Ink4a, diminuiu a patologia associada a diferentes doenças do envelhecimento, incluindo a degeneração do disco intervertebral. Assim, caracterizamos pela primeira vez a evolução da senescência nas células do disco intervertebral de ratinhos jovens e envelhecidos e avaliamos o fenótipo de ratinhos envelhecidos p16Ink4a “condition knockout” (cKO). Adicionalmente, exploramos o potencial terapêutico da combinação de Dasatinib (D) e Quercetina (Q), na diminuição de células senescentes no disco e na prevenção da degeneração do mesmo durante o envelhecimento. Estes resultados sugerem que a regulação da senescência é importante na promoção e manutenção da homeostasia das células do disco, e que pode ser usado com alvo terapêutico para a prevenção e tratamento da degeneração do disco durante o envelhecimento.The intervertebral disc is a critical structure of the spinal column, responsible for mechanical loads and flexibility. Importantly, disc extracellular matrix (ECM) is complex and highly contributes to the biomechanical properties of the disc. However, it is still not well understood due to its complexity and lack of experimental models. Disc degeneration presents a broad spectrum of clinically degenerative phenotypes during aging, such as fibrosis, calcification, herniation, and mild degeneration. Moreover, the interaction between genetic background and aging, degenerative outcomes has not been established. Hence, we characterized the spinalphenotype of 3 mouse strains with varying cartilage-regenerative potential at 6- and 23-months: C57BL/6 (BL6), LG/J, and SM/J. Interestingly, each strain presented an unique aging phenotype and transcriptome modulation during intervertebral disc aging. LG/J mice showed a high prevalence of dystrophic disc calcification in caudal discs, while SM/J evidenced accelerated degeneration compared to BL6, with cellular disorganization, cell loss together, and fibrosis of the NP. These studies suggested that disc aging and degeneration depends on the genetic background and involves changes in various molecular pathways, which might help to explain the diverse phenotypes seen during disc disease. Additionally, recent studies have shown that senescence may play a role in age-related disc degeneration and matrix catabolism in humans and mouse models. Clearance of p16Ink4a, one of the main markers of cell senescence, positive cells reduce the degenerative phenotype in age-associated diseases, including disc degeneration. Therefore, we first characterized the senescence status of discs in young and old mice and accessed the degeneration status of aged p16Ink4a conditional knockout mice. Additionally, we explored the potential of Dasatinib and Quercetin combination in targeting senescence in disc compartment and preventing progression of the disease with age. These results suggested that senescence plays an essential role in governing disc homeostasis during aging and can be used as a therapeutic target to treat disc disease.Financial support for this PhD work was provided by Fundação para a Ciência e a Tecnologia (FCT) fellowship (PD/BD/128077/2016) from MD/PhD Program of the University of Minho. Additionally, this study was supported by the grants from the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) R01AR055655, R01AR064733 and R01AR074813 to Makarand V. Risbud