Agradecemos C. Park para ler o manuscrito. Este trabalho foi apoiado pelo NIAMS sob Award Número K01AR061434 e uma Sociedade Fellowship Award Leukemia &amp; Lymphoma (5127-09) para DP e doações dos Institutos Nacionais de Saúde para CPL e DTS O conteúdo é de responsabilidade exclusiva de seus autores e não representam, necessariamente, a posição oficial do National Institutes of Health.

1. Ratos e pré-condicionamento Nota: Todos os animais foram mantidos em condições livres de patógenos e todos os protocolos foram aprovados pelo Animal Care e Use Comitê Institucional (IACUC) no Hospital Geral de Massachusetts. Todos cirurgia deve ser realizada sob condição estéril utilizando equipamento estéril autoclavada. Mx1-Cre 15, Rosa26-loxP-stop-loxP-EYFP (Rosa-YFP) e Rosa26-loxP-stop-loxP-tdTomato (Rosa-tomate), foram comprado a partir de Jackson Laboratories. ratinhos osteocalcina-GFP foram fornecidos pelo Dr. Henry Kronenberg. Para a análise quantitativa de migração OSPC e proliferação in vivo, foram utilizados Mx1-Cre + Rosa-YFP + (única cor-repórter) ratos. Para acompanhamento mais detalhado de sua diferenciação em osteoblastos Osteocalcin + amadurecer, usamos trigenic Mx1-Cre + Rosa-tomate + OCN-GFP +ratinhos. <ol><li> Para marcar células Mx1 + in vivo, preparar ácido polyinosinic-polycytidylic (PIPC, 2,5 mg / ml) em solução de PBS estéril e injectar 200 ul de 20 g de Mx1-Cre + repórter rato intraperitonealmente uma vez em dias alternados durante 10 dias. </li><li> Para eliminar as células hematopoiéticas Mx1 + residentes, irradiar ratinhos com uma dose única de 9,5 Gy. Após 24 horas de irradiação, tipo selvagem células da medula óssea transplantados (1x10 6 células / rato) por via intravenosa 16. Nota: Como as células Mx1-induzidos incluem células hematopoiéticas e osteoclastos derivados de hematopoiéticas, a eliminação de Mx1 + células hematopoiéticas melhora a qualidade de imagem e quantificação de células Mx1 + osteogênicas. </li><li> Após o transplante de medula óssea, para monitorizar os animais quatro a seis semanas, a fim de alcançar um repovoamento bem sucedida de células de medula do dador. </li></ol> 2. Mousar Preparação <ol><li> Anestesiar rato por injecção intraperitoneal de 50 ul de cetamina / xilazina (100 mg / kg, xilazina 12 mg / kg de peso corporal), utilizando um procedimento IACUC-aprovado. Nota: A duração do efeito pode ser prorrogado por uma dose adicional de cetamina / xilazina. </li><li> Determinar se o mouse está totalmente anestesiado pela falta de resposta aos pés e / ou pitadas de cauda. (Opcional) Quando o mouse é anestesiado, garantir uma máscara de gás isoflurano sobre o nariz gravando. Ajuste o nível de isoflurano para garantir que o animal está completamente sedada. </li><li> Prenda o cabelo do couro cabeludo usando um aparador elétrico ou tesoura pequena. Retire os pedaços de cabelo e esterilizar a pele exposta com 70% de algodão embebido em álcool. Aplique gel lacrimogêneo para evitar a desidratação da córnea. </li></ol> 3. Lesões Microfratura <ol><li> Depois de garantir que o animal está totalmente sedado, fazer uma única incisão inversa em forma de L para abrir a pele do couro cabeludo. Resumidamente, fazer uma primeira incisão (menor que 1 cm) starting de uma orelha a outra orelha. Vire ~ 60 ° e continuar a incisão em direção ao nariz até ~ 2-3 mm de distância do nariz. Nota: Esta incisão minimiza a formação de tecidos cicatriciais acima da área a ser trabalhada. </li><li> Separe os retalhos de pele puxando para os lados com uma pinça. Ambos os ossos frontais e da intersecção das suturas sagital e coronal deve ser claramente exposto. </li><li> Limpe a superfície aberta por lavagem com PBS estéril e limpeza suave com gaze ou cotonetes estéreis até que todos os pêlos residuais são eliminados. </li><li> Para gerar microfraturas no calvário, segure a cabeça do rato com uma mão e uma agulha 30 G com a outra mão. Faça um micro-punção (~ ferida diâmetro 0,2 milímetros com &lt;1mm de profundidade) no osso frontal esquerdo perto do cruzamento das suturas sagital e coronal, inserindo a ponta de uma agulha 30 G com uma pressão suave e torcer movimento. Cuidado: é essencial para evitar a agulha de penetrar no cérebro, minimizando assim bleeding e dano tecidual. </li><li> Mudar para uma agulha maior (20 ou 25 G) e alargar o buraco punção torcendo a agulha (~ 0,5 milímetros de diâmetro). </li><li> Repita o passo 3,3-3,5 para gerar segunda punção no osso frontal contralateral. </li><li> Limpe os pontos feridos pela queda contínua da PBS até o sangramento parar. Nota: sangramento em locais de fratura irá interferir com a imagem apropriada e induzir a formação de cicatriz. </li><li> Aplicar uma gota de solução salina fisiológica estéril ou 2% Methocel no crânio para evitar a secagem. Nota: Não permita que a superfície do crânio para secar, o que irá reduzir a nitidez da imagem. É importante utilizar uma quantidade suficiente de gel ou solução salina para cobrir a área. </li></ol> 4. Imagens intravital <ol><li> (Opcional) Antes de imagiologia, alguns ratos foram injectados com um corante vascular para visualizar os vasos sanguíneos. Injetar retro-orbital com 20 l de Qtracker não-alvo 705 (2 mM solução em 50 mM de tampão borato) diluído em 80 ml de PBS. Use uma seringa de insulina para minimizar o desperdício de reagentes. Se o sinal não é suficientemente brilhante, valores adicionais podem ser necessárias. </li><li> Ligue o scanner. Nota: Um scanner a laser com base em polígono permite a aquisição simultânea de múltiplos canais de imagem a uma taxa de vídeo (30 quadros por segundo). Digitalização taxa vídeo é muito importante para a imagem do animal vivo. </li><li> Ligue o laser multi-fotão (femto-segundo titânio: laser de safira). Defina o comprimento de onda de 880 nm e ajustar a potência de segunda geração de imagens harmônico (440 nm) do osso. Para GFP e tdTomato excitação, ligue o 491 nm e 561 lasers de estado sólido nm. </li><li> Ligue a PMT (tubo fotomultiplicador) detectores para cada sinal (± 20 nm filtro passa-banda 435 para geração de segundo harmônico, a ± 19 nm filtro passa-banda de 528 para GFP, e 590 ± 20 para tdTomato.) (Opcional) Use um 638 nm laser de hélio-neon e um PMT com ± 27,5 nm filtro passa-banda de 695 para detectar Qtracker sinal 705. </li><li> Placenviar um e o animal em um eixo XYZ estágio do microscópio motorizado. Mantenha o mouse na posição mais confortável com uma almofada de aquecimento elétrico para ajudar a manter a temperatura do corpo (o que reduz o risco de perda de animais). Use fita adesiva para segurar o mouse para minimizar o movimento durante o exame e para manter a área com imagens o mais horizontal possível. </li><li> Aplicar uma gota de quente 2% de gel de metilcelulose ou de solução salina fisiológica sobre o crânio para evitar a secagem. Coloque uma tampa de vidro sobre a área de imagem. Use uma lente de baixa ampliação (30x objetiva de imersão em água com 0,9 NA) para digitalizar o calvária. </li><li> Usando o controlador dos eixos XYZ, encontrar a superfície de calvária de detectar o sinal de SHG a partir de ossos e identificar um local de marco fundamental, tal como a intersecção entre as suturas sagital e coronal suturas. Adquirir uma imagem e gravar as coordenadas XYZ. </li><li> Continue a procurar o local da lesão, observando sinais de SHG e fluorescência. </li><li> Quando os sites de ferimento ou da região of interesse são encontrados, adquirir uma imagem do melhor plano focal contendo sinais de fluorescência SHG e das células de interesse. Salvar coordenadas XYZ ea distância até à intersecção das suturas sagital e coronal para definir sua localização exacta para as próximas rodadas da imagem. </li><li> Para a coleta de estruturas 3D celulares e osso de lesão fratura, gravar imagens por Z-stacks (2-5 intervalo mM) com ~ 100 mm de profundidade a partir da superfície do osso endosteal. Após a conclusão da imagem de um lado da lesão, repetir processo de imagem para sites de lesões próximas. </li></ol> Procedimentos 5. Operação POST <ol><li> Após a imagiologia, enxaguar local de imagem com solução salina estéril para remover o gel de metilcelulose a partir do crânio. Usando uma mecha de algodão estéril, aplicar uma pequena quantidade de pomada com antibiótico triplo na superfície. Cubra os retalhos de pele e re-fechar o couro cabeludo por sutura cirúrgica. Isto pode ser feito com um fio de sutura hipoalergénico (poliglactina absorvível sutura) e 5-0 tamanho da agulha de sutura. Atenção: A boa técnica de sutura minimiza a formação de cicatriz. </li><li> Tratar todos os animais com injeção IP de 0,05-0,1 mg / kg de buprenorfina cada 12 horas para 48 horas após a cirurgia. Manter os animais em uma câmara de recuperação quente até que recuperar a consciência suficiente. Após 3 a 5 dias, a reabertura da sutura e repita os passos de imagens intravital para acompanhar a mudança celular durante a cicatrização da fratura. </li></ol>

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Os autores declaram que não têm interesses financeiros competitivos.

O regulamento de células-tronco do esqueleto pode ser de grande importância para a definição de melhores métodos para alcançar a regeneração óssea. Imagens quantitativas e seqüencial no nível celular tem sido tecnicamente desafiador. Embora o modelo de fractura de ossos longos do rato tem sido amplamente utilizado e adequado para estudos biomecânicos 17, a sua localização de tecidos profundos, fractura tamanho desigual, danos nos tecidos moles, e a aplicação de fixadores de estabilização tê...

Doenças ósseas degenerativas e perda óssea relacionada à idade levando a um alto risco de fratura osteoporótica tornou-se um grande desafio para a saúde pública 1. Manutenção do osso é controlado pelos osteoblastos de formação óssea e os osteoclastos de reabsorção óssea. Defeitos de formação de células ósseas são uma das principais causas de perda óssea relacionada à idade e doenças ósseas degenerativas 2,3. Embora extensa pesquisa centrou-se na melhoria da consolidação da fratura, a descoberta de medicamentos confiáveis ​​para curar doenças ósseas degenerativas e para reverter a fraqueza de fraturas osteoporóticas continua a ser uma questão importante. Assim, o estudo da fonte de células formadoras de osso e de seus mecanismos de controle na regeneração óssea e reparação oferece uma nova visão para melhorar a regeneração óssea e reverter doenças perda óssea. A existência de células mesenquimais multipotentes da medula óssea foi proposto com base na identificação de populações clonogénicos que poderia diferenteIATE em osteogênico, linhagens adipogénicos e condrogênicas ex vivo 4. Recentemente, vários estudos têm relatado que as células-tronco do esqueleto / mesenquimais (SSC / CTM) são uma fonte natural de osteoblastos e são fundamentais para a renovação óssea, remodelação e reparo de fratura 5,6 . Além disso, nosso estudo de rastreamento de linhagem revelou que osteoblastos maduros têm uma meia-vida inesperadamente curto (~ 60 dias) e são continuamente reabastecidos pelas suas células tronco / progenitoras em ambas as condições homeostáticas e reparo de fratura normais 6. No entanto, a identidade in vivo de células estaminais e como tal as células reagem para fracturar lesão e fornecimento de células formadoras de osso não são claras. Portanto, é importante desenvolver um método que é capaz de analisar a migração, proliferação e diferenciação das CSCs endógenos / MSCs em circunstâncias fisiológicas. Reparo de fratura é um processo multi-celular e dinâmica regulada por um conjunto de complexocitoquinas e factores de crescimento de 7. A abordagem mais popular para estudos de fratura é a utilização de um modelo animal com fratura de ossos longos e analisar ossos por seccionamento do osso e as técnicas de imunofluorescência 8-10. Este processo de reparação pode ser monitorado por várias técnicas de imagem, incluindo micro-CT 11, de infravermelho próximo de fluorescência 12, e imagens quimiluminescência 13. No entanto, cada técnica tem algumas limitações e não houve nenhuma maneira eficaz de monitorar a função SSC / MSC no nível celular in vivo. Recentemente, confocal / dois fotões microscopia intravital tem sido desenvolvido e usado para detectar células cancerosas transplantadas e células-tronco hematopoiéticas no contexto do seu microambiente da medula óssea, mesmo na resolução de uma única célula em animais vivos 14. Ao combinar essa tecnologia com uma série de modelos de rastreamento de linhagem, fomos capazes de definir que as células-tronco osteogênico / progenitoras podem ser geneticamente marcado por ac transitóriavação da resistência myxovirus -1 (Mx1) promotor e progenitores induzida-MX1 pode manter a maioria dos osteoblastos maduros ao longo do tempo, mas não participam na geração de condrócitos no rato adulto 6. Além disso, demonstrou-se que OSPCs Mx1 marcadas fornecer a maior parte dos novos osteoblastos em fratura cura 6. Aqui, utilizando modelos de rastreamento osteo-linhagem e microscopia intravital, um protocolo é fornecido para definir os in vivo cinética de células-tronco + / progenitoras osteogênicas Mx1 no reparo de fratura. Este protocolo oferece imagens seqüenciais para acompanhar a deslocalização de osteogênicas tronco / progenitoras em sítios de fratura e a medição quantitativa de expansão osteoprogenitoras no processo de reparo precoce. Esta abordagem pode ser útil em vários contextos, incluindo a avaliação de candidatos terapêuticos para melhorar a reparação do osso.

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			<td height="63" style="height:63px;width:216px;">C57BL/6J (H-2b)</td>
			<td style="width:140px;">Jackson Laboratories (Bar Harbor, ME)</td>
			<td style="width:195px;">000664</td>
			<td style="width:279px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ketamine Hydrochloride Injection</td>
			<td style="width:140px;">Bionichepharma</td>
			<td style="width:195px;">67457-001-10</td>
			<td>&#160;Vial size: 10 ml (50 mg/ml)</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Xylazine Sterile Solution</td>
			<td style="width:140px;">Lloyd Inc.</td>
			<td style="width:195px;">NADA# 139-236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Buprenorphine Hl</td>
			<td style="width:140px;">BEDFORD LAB</td>
			<td style="width:195px;">NDC 55390-100-10</td>
			<td>Vial: 0.3 mg/ml, Doses: 0.05-0.1 mg/kg</td>
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			<td height="21" style="height:21px;width:216px;">DPBS, 1X</td>
			<td style="width:140px;">CORNING cellgro</td>
			<td style="width:195px;">21-031-CV</td>
			<td></td>
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			<td height="42" style="height:42px;width:216px;">Alcohol Prep Pads (70% Isopropyl alcohol)</td>
			<td style="width:140px;">Kendall WEBCOL</td>
			<td style="width:195px;">5110</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Fine Surgical Scissor</td>
			<td style="width:140px;">F.S.T</td>
			<td style="width:195px;">14568-09</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Extra fine Forceps</td>
			<td style="width:140px;">F.S.T</td>
			<td style="width:195px;">11150-10</td>
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			<td height="21" style="height:21px;width:216px;">VICRYL*Plus Suture</td>
			<td style="width:140px;">Ethicon</td>
			<td style="width:195px;">VCP490G</td>
			<td></td>
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			<td height="42" style="height:42px;width:216px;">Qtracker 705 non-targeted quantum dot</td>
			<td style="width:140px;">Invitrogen</td>
			<td style="width:195px;">Q21061</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Methocel 2%</td>
			<td style="width:140px;">OmmiVision</td>
			<td style="width:195px;"></td>
			<td></td>
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			<td height="42" style="height:42px;width:216px;">pIpC (Polyinosinic-polycytidylic acid)&#160;&#160;</td>
			<td style="width:140px;">Sigma</td>
			<td style="width:195px;">P0913-50MG</td>
			<td>100 &#956;l (2.5 mg/ml in PBS) for 10 g of mouse</td>
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			<td height="42" style="height:42px;width:216px;">Mai Tai Tunable Ultrafast Lasers</td>
			<td style="width:140px;">Spectra Physics</td>
			<td style="width:195px;"></td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dual Calypso 491 + 532 nm DPSS laser</td>
			<td style="width:140px;">Cobolt AB</td>
			<td style="width:195px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Radius-635 HeNe laser</td>
			<td style="width:140px;">Coherent</td>
			<td style="width:195px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

sequential in vivo imaging of osteogenic stem/progenitor cells during fracture repair

Óssea vira continuamente e é altamente regenerativo após lesão. Células tronco / progenitoras osteogênicas têm sido a hipótese de existir, mas em demonstração vivo dessas células só recentemente foi atingido. Aqui, são fornecidos in vivo técnicas de imagem para investigar o papel das células-tronco / progenitoras osteogênicas endógenos (OSPCs) e seus descendentes na reparação óssea. Usando osteo-linhagem de células traçando modelos e imagens intravital de microfraturas induzidas no osso calvária, OSPCs podem ser observados diretamente durante os primeiros dias após a lesão, em que ocorrem eventos críticos no processo de reparo precoce. Sites de lesão pode ser trabalhada sequencialmente revelando que OSPCs mudar para a lesão, aumentam em número e diferenciação em osteoblastos do osso formando. Estes métodos oferecem um meio de investigar o papel dos reguladores moleculares Stem Cell-intrínsecos e extrínsecos para regeneração óssea e reparo.

<html> O modelo de fratura de ossos longos estabilizada tem sido popular em estudos de fratura. No entanto, ossos longos ou grandes modelos de fratura causar danos nos tecidos múltiplos e, portanto, têm uma limitação na medição quantitativa da função das células do osso. Nós desenvolvemos uma lesão minimamente invasiva (menos de 1 mm de diâmetro, com pouca ou nenhuma invasão na dura-máter) sobre os ossos de calvária frontais com agulha de perfuração (Figuras 1A-1C). Nós escolhemos uma v...

Watch this Scientific Journal Video about Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair at JoVE.com

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

A medida quantitativa da função progenitor óssea na consolidação da fratura requer alta resolução tecnologia de imagens em série. Aqui, os protocolos são fornecidos para o uso de microscopia intravital e rastreamento osteo-linhagem sequencialmente imagem e quantificar a migração, proliferação e diferenciação de células-tronco / progenitoras osteogênicas endógenos no processo de reparação de fratura óssea.

Seqüente<em&gt; In vivo</em&gt; Imagem de osteogênicas células-tronco / progenitoras durante o reparo de fratura

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in ...

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Research

JoVE Journal

Medicine

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

10.4K visualizações.  Harvard Stem Cell Institute.  A medida quantitativa da função progenitor óssea na consolidação da fratura requer alta resolução tecnologia de imagens em série. Aqui, os protocolos são fornecidos para o uso de microscopia intravital e rastreamento osteo-linhagem sequencialmente imagem e quantificar a migração, proliferação e diferenciação de células-tronco / progenitoras osteogênicas endógenos no processo de reparação de fratura óssea. 

Vídeo: Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

In vivo Dual Substrate Bioluminescent Imaging

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate since the advent of in vivo bioluminescent imaging technologies 1-3. Indeed, the ability to locate and quantify tumor growth longitudinally in a single cohort of animals to completion of the study as opposed to sacrificing individual groups of animals at specific assay times has revolutionized how researchers investigate breast cancer metastasis. Unfortunately, current methodologies preclude the real-time assessment of critical changes that transpire in cell signaling systems as breast cancer cells (i) evolve within primary tumors, (ii) disseminate throughout the body, and (iii) reinitiate proliferative programs at sites of a metastatic lesion. However, recent advancements in bioluminescent imaging now make it possible to simultaneously quantify specific spatiotemporal changes in gene expression as a function of tumor development and metastatic progression via the use of dual substrate luminescence reactions. To do so, researchers take advantage for two light-producing luciferase enzymes isolated from the firefly (Photinus pyralis) and sea pansy (Renilla reniformis), both of which react to mutually exclusive substrates that previously facilitated their wide-spread use in in vitro cell-based reporter gene assays 4. Here we demonstrate the in vivo utility of these two enzymes such that one luminescence reaction specifically marks the size and location of a developing tumor, while the second luminescent reaction serves as a means to visualize the activation status of specific signaling systems during distinct stages of tumor and metastasis development. Thus, the objectives of this study are two-fold. First, we will describe the steps necessary to construct dual bioluminescent reporter cell lines, as well as those needed to facilitate their use in visualizing the spatiotemporal regulation of gene expression during specific steps of the metastatic cascade. Using the 4T1 model of breast cancer metastasis, we show that the in vivo activity of a synthetic Smad Binding Element (SBE) promoter was decreased dramatically in pulmonary metastasis as compared to that measured in the primary tumor 4-6. Recently, breast cancer metastasis was shown to be regulated by changes within the primary tumor microenvironment and reactive stroma, including those occurring in fibroblasts and infiltrating immune cells 7-9. Thus, our second objective will be to demonstrate the utility of dual bioluminescent techniques in monitoring the growth and localization of two unique cell populations harbored within a single animal during breast cancer growth and metastasis.

In vivo Dual Substrate Bioluminescent Imaging

Herein we describe the methods to construct, visualize, and quantify the bioluminescent reactions of both firefly and renilla luciferase enzymes expressed in metastatic breast cancer cells during their growth and metastasis in vivo.

In vivo dupla Substrato Bioluminescent imagem

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate ...

Medicina

3D-Neuronavigation In Vivo Through a Patient's Brain During a Spontaneous Migraine Headache

A growing body of research, generated primarily from MRI-based studies, shows that migraine appears to occur, and possibly endure, due to the alteration of specific neural processes in the central nervous system. However, information is lacking on the molecular impact of these changes, especially on the endogenous opioid system during migraine headaches, and neuronavigation through these changes has never been done. This study aimed to investigate, using a novel 3D immersive and interactive neuronavigation (3D-IIN) approach, the endogenous &#181;-opioid transmission in the brain during a migraine headache attack in vivo. This is arguably one of the most central neuromechanisms associated with pain regulation, affecting multiple elements of the pain experience and analgesia. A 36 year-old female, who has been suffering with migraine for 10 years, was scanned in the typical headache (ictal) and nonheadache (interictal) migraine phases using Positron Emission Tomography (PET) with the selective radiotracer [11C]carfentanil, which allowed us to measure &#181;-opioid receptor availability in the brain (non-displaceable binding potential - &#181;OR BPND). The short-life radiotracer was produced by a cyclotron and chemical synthesis apparatus on campus located in close proximity to the imaging facility. Both PET scans, interictal and ictal, were scheduled during separate mid-late follicular phases of the patient's menstrual cycle. During the ictal PET session her spontaneous headache attack reached severe intensity levels; progressing to nausea and vomiting at the end of the scan session. There were reductions in &#181;OR BPND in the pain-modulatory regions of the endogenous &#181;-opioid system during the ictal phase, including the cingulate cortex, nucleus accumbens (NAcc), thalamus (Thal), and periaqueductal gray matter (PAG); indicating that &#181;ORs were already occupied by endogenous opioids released in response to the ongoing pain. To our knowledge, this is the first time that changes in &#181;OR BPND during a migraine headache attack have been neuronavigated using a novel 3D approach. This method allows for interactive research and educational exploration of a migraine attack in an actual patient's neuroimaging dataset.

3D-Neuronavigation In Vivo Through a Patient&#039;s Brain During a Spontaneous Migraine Headache

In this study, the authors report for the first time a novel 3D-Immersive &#38; Interactive Neuronavigation (3D-IIN) through the impact of a spontaneous migraine headache attack in the &#956;-opioid system of a patient's brain in vivo.

3D-neuronavegação<em&gt; In Vivo</em&gt; Através do cérebro de um paciente durante uma espontânea enxaquecas

A growing body of research, generated primarily from MRI-based studies, shows that migraine appears to occur, and possibly endure, due to the alteration ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

In vivo 19 F MRI para Rastreamento celular

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with distinct functions and gene expression profiles. The roles of microglial activation in health, injury and disease remain incompletely understood due to their dynamic and complex regulation in response to changes in their microenvironment. Thus, it is critical to non-invasively monitor and analyze changes in microglial activation over time in the intact organism. In vivo studies of microglial activation have been delayed by technical limitations to tracking microglial behavior without altering the CNS environment. This has been particularly challenging during chronic neurodegeneration, where long-term changes must be tracked. The retina, a CNS organ amenable to non-invasive live imaging, offers a powerful system to visualize and characterize the dynamics of microglia activation during chronic disorders.
This protocol outlines methods for long-term, in vivo imaging of retinal microglia, using confocal ophthalmoscopy (cSLO) and CX3CR1GFP/+ reporter mice, to visualize microglia with cellular resolution. Also, we describe methods to quantify monthly changes in cell activation and density in large cell subsets (200-300 cells per retina). We confirm the use of somal area as a useful metric for live tracking of microglial activation in the retina by applying automated threshold-based morphometric analysis of in vivo images. We use these live image acquisition and analyses strategies to monitor the dynamic changes in microglial activation and microgliosis during early stages of retinal neurodegeneration in a mouse model of chronic glaucoma. This approach should be useful to investigate the contributions of microglia to neuronal and axonal decline in chronic CNS disorders that affect the retina and optic nerve.

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia activation and microgliosis are key responses to chronic neurodegeneration. Here, we present methods for in vivo, long-term visualization of retinal CX3CR1-GFP+ microglial cells by confocal ophthalmoscopy, and for threshold and morphometric analyses to identify and quantify their activation. We monitor microglial changes during early stages of age-related glaucoma.

In Vivo Dinâmica da Retinal microglia Ativação Durante Neurodegeneration: confocal oftalmoscópico Imagem e morfometria celular no mouse Glaucoma

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with ...

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest improved outcome measures in both experimental and clinical studies. Although the BMMSC are implanted into the tendon lesion in large numbers (usually 10 - 20 million cells), only a relatively small number survive (&#60;10%) although these can persist for up to 5 months after implantation. This appears to be a common observation in other species where BMMSC have been implanted into other tissues and it is important to understand when this loss occurs, how many survive the initial implantation process and whether the cells are cleared into other organs. Tracking the fate of the cells can be achieved by radiolabeling the BMMSC prior to implantation which allows non-invasive in vivo imaging of cell location and quantification of cell numbers.
This protocol describes a cell labeling procedure that uses Technetium-99m (Tc-99m), and tracking of these cells following implantation into injured flexor tendons in horses. Tc-99m is a short-lived (t1/2 of 6.01 hr) isotope that emits gamma rays and can be internalized by cells in the presence of the lipophilic compound hexamethylpropyleneamine oxime (HMPAO). These properties make it ideal for use in nuclear medicine clinics for the diagnosis of many different diseases. The fate of the labeled cells can be followed in the short term (up to 36 hr) by gamma scintigraphy to quantify both the number of cells retained in the lesion and distribution of the cells into lungs, thyroid and other organs. This technique is adapted from the labeling of blood leukocytes and could be utilized to image implanted BMMSC in other organs.

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

This protocol describes the radiolabeling of equine mesenchymal stem cells and their implantation into tendon injuries in the horse in order to determine cell survival and tissue distribution using gamma scintigraphy.

In Vivo Imaging and Rastreamento de Tecnécio-99m Rotulado de Medula Óssea Células-Tronco Mesenquimais em Equine Tendinopatia

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Estudar o cancro do pâncreas Stem características celulares para desenvolver estratégias Novo tratamento

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

Depleção de células do rato de xenoenxertos de tumor humano Melhora Significativamente Análise Downstream de células alvo

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The amount of the different tissues in the callus provides important information on the fracture healing progress. Available in vivo techniques to longitudinally monitor the callus tissue development in preclinical fracture-healing studies using small animals include digital radiography and &#181;CT imaging. However, both techniques are only able to distinguish between mineralized and non-mineralized tissue. Consequently, it is impossible to discriminate cartilage from fibrous tissue. In contrast, magnetic resonance imaging (MRI) visualizes anatomical structures based on their water content and might therefore be able to noninvasively identify soft tissue and cartilage in the fracture callus. Here, we report the use of an MRI-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice. The experiments demonstrated that the fixator and a custom-made mounting device allow repetitive MRI scans, thus enabling longitudinal analysis of fracture-callus tissue development.

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

The evaluation of tissue development in the fracture callus during endochondral bone healing is essential to monitor the healing process. Here, we report the use of a magnetic resonance imaging (MRI)-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice.

Na Vivo Avaliação do desenvolvimento de calo de fratura durante a consolidação óssea em ratos usando um dispositivo de osteossíntese compatível com MRI para o fêmur de Mouse

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The ...

Middle Cerebral Artery Occlusion Allowing Reperfusion via Common Carotid Artery Repair in Mice

The ischemic stroke is a major cause of adult long-term disability and death worldwide. The current treatments available are limited, with only tissue plasminogen activator (tPA) as an approved drug treatment to target ischemic strokes. Current research in the field of ischemic stroke focuses on better understanding the pathophysiology of stroke, to develop and investigate novel pharmaceutical targets. Reliable experimental stroke models are crucial for the progression of potential treatments. The middle cerebral artery occlusion (MCAO) model is clinically relevant and the most frequently used surgical model of ischemic stroke in rodents. However, the outcomes of this model, such as lesion volume, are associated with high levels of variability, particularly in mice. The alternative MCAO model described here allows the reperfusion of the common carotid artery (CCA) and the increased perfusion of the middle cerebral artery (MCA) territory, using a tissue pad with fibrinogen-based sealant to repair the vessel, and the improved welfare of the mice by avoiding external carotid artery (ECA) ligation. This reduces the reliance on the Circle of Willis, which is known to be highly anatomically variable in mice. Representative data show that using this alternative surgical approach decreases the variability in lesion volumes between the traditional MCAO approach and the alternative approach described here.

Intraluminal filament occlusion of the middle cerebral artery is the most frequently used in vivo model of experimental stroke in rodents. An alternative surgical approach to allow common carotid artery repair is performed here, which allows the reperfusion of the common carotid artery and a full reperfusion to the middle cerebral artery territory.

Oclusão de artéria Cerebral média, permitindo a reperfusão através de reparação de artéria carótida comum em ratos

The ischemic stroke is a major cause of adult long-term disability and death worldwide. The current treatments available are limited, with only tissue ...

A Simplified Stepwise Approach to Echo Guidance during Percutaneous Mitral Valve Repair

Percutaneous transcatheter edge-to-edge reconstruction of the mitral valve is a safe and well-established therapy for severe symptomatic mitral regurgitation in patients with high surgical risk. Echocardiographic guidance in addition to fluoroscopy is the gold standard and should be performed using a standardized technique.
This article lays out our reproducible step by step echocardiographic guide including views, measurements as well as highlighting possible difficulties that may arise during the procedure.
This article provides detailed and chronological echocardiographic views for each step of the procedure, especially preferences between 2D and 3D imaging. If needed, pulse wave, continuous wave and color doppler measurements are described. Furthermore, as there are no official recommendations for the quantification of mitral regurgitation during the percutaneous edge-to-edge-repair procedure, advice is also included for echocardiographic quantification after grasping the mitral leaflets and after device deployment. In addition, the article deals with important echocardiographic views to prevent and deal with possible complications during the procedure.
Echocardiographic guidance during transcatheter mitral valve repair is mandatory. A structured approach improves the collaboration between interventionist and imager and is indispensable for a safe and effective procedure.

This protocol presents in detail how to perform real-time echocardiographic guidance during transcatheter mitral valve repair. The fundamental views and the necessary measurements are described for each stage of the procedure.