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

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

On different scales: the architecture of Hospital- Colônia Rovisco Pais from the perspective of doctor Fernando Bissaya Barreto

Em 1938, o médico português Fernando Bissaya Barreto promoveu a construção de um ‘internato de leprosos’ no centro de Portugal, afastado dos grandes centros populacionais, mas de fácil acesso a todo o país. Inaugurado em 1947, o Hospital-Colônia Rovisco Pais segue o modelo de um hospitalasilo- colônia, sendo simetricamente segmentado por pavilhões iguais em número e características para os dois sexos. Seguindo uma lógica disciplinar e não de exclusão, o plano urbanístico, o desenho dos edifícios e o próprio mobiliário e decoração são configurados, sob a influência direta de Bissaya Barreto, como instrumentos de intervenção sobre o corpo físico e moral dos doentes de Hansen, mas também, e em outra escala, de controle e modificação da sociedade portuguesa no seu conjunto.In 1938, Portuguese physician Fernando Bissaya Barreto spearheaded the creation of a ‘nursing home for lepers’ in the center of Portugal, away from big towns and cities, but still accessible from any part of the country. Opened in 1947, Hospital- Colônia Rovisco Pais followed the model of a colony/hospital/hospice, and was divided symmetrically into buildings of equal features and numbers for both sexes. According to a disciplinary, non-exclusionary rationale, the urban design, building design and furniture and fittings were conceived, under the direct influence of Bissaya Barreto, as instruments for intervention in the physical and moral bodies of the patients, and also, on a different scale, for the control and modification of Portuguese society as a whole

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

Milho verde e feijão-caupi cultivados em consórcio sob diferentes lâminas de irrigação e doses de fósforo

O objetivo deste trabalho foi avaliar a resposta de milho verde (Zea mays) e de feijão-caupi (Vigna unguiculata), cultivados em consórcio, a lâminas de irrigação e doses de fósforo. Os experimentos foram realizados em 2008 e 2009, em delineamento de blocos ao acaso, com 25 tratamentos e quatro repetições. Os tratamentos consistiram de cinco lâminas de irrigação, a 70, 110, 140, 180 e 220% da evapotranspiração da cultura, e de cinco doses de P2O5 a 0, 50, 100, 150 e 200% da dose de P recomendada. O milho foi semeado no espaçamento 0,80x0,40 m, e o feijão-caupi foi semeado dentro das linhas entre as plantas de milho. Não houve efeito das doses de P2O5; porém, a resposta às lâminas de irrigação foi quadrática em milho e linear em feijão-caupi. As máximas produtividades técnicas de espigas de milho verde com palha (10,76 Mg ha-1) e sem palha (7,62 Mg ha-1) foram obtidas com a lâmina de 530 mm, intermediária às lâminas referentes a 180 e 220% da evapotranspiração da cultura. A maior produtividade de grãos verdes de feijão-caupi (3,40 Mg ha-1) foi obtida com a maior lâmina de água aplicada, de 644 mm

Characterisation of microbial attack on archaeological bone

As part of an EU funded project to investigate the factors influencing bone preservation in the archaeological record, more than 250 bones from 41 archaeological sites in five countries spanning four climatic regions were studied for diagenetic alteration. Sites were selected to cover a range of environmental conditions and archaeological contexts. Microscopic and physical (mercury intrusion porosimetry) analyses of these bones revealed that the majority (68%) had suffered microbial attack. Furthermore, significant differences were found between animal and human bone in both the state of preservation and the type of microbial attack present. These differences in preservation might result from differences in early taphonomy of the bones. © 2003 Elsevier Science Ltd. All rights reserved