A.S. was partially supported by grants from the National Institutes of Health (NIH) National Institute of Dental and Craniofacial Research (5R03DE021741-02) and the National Cancer Institute (1K08CA154963-01A1).&#160;
The Howard Hughes Medical Institute provided funding for M.L. through the HHMI Medical Research Fellows Program.

1. A incubação do ovo <ol><li> Obter 8 dias de idade ovos isentos de agentes patogénicos específicos embrionados. </li><li> Coloque os ovos na rotação bandeja de ovos, estampadas extremidades voltadas para cima, e coloque a bandeja girando dentro de uma incubadora de ovos. Incubar os ovos durante 48 horas a 36 ° C e 50% de humidade. </li></ol> 2. Deixar cair o CAM e abrindo os ovos <ol><li> Recolher os ovos da incubadora. Coloque os ovos na prateleira dos ovos, carimbado lado de cima, e trazer à capela de fluxo laminar. </li><li> Desligue as luzes da sala e do capô. </li><li> Segurar o fim visado de ovo levemente para o candler ovo para expor a vasculatura de CAM, bem como o saco de ar. NOTA: O candler ovo é uma fonte de luz usado para visualizar o desenvolvimento do embrião e do saco de ar associado e vasculatura. </li><li> Coloque uma marca de lápis entre dois grandes vasos sanguíneos. </li><li> Ligar as luzes e usar um pino estéril para fazer um orifício na ponta do ovo por cima do saco de ar (final estampado) e um furo na pemarca NCIL. Não empurre o pino do impulso durante todo o tempo; um furo de cerca de 3 mm de profundidade será normalmente suficiente. </li><li> Apaga as luzes. Squeeze bulbo segurança, pressionando a extremidade aberta firmemente contra o buraco acima do saco de ar. Aplicar sucção para puxar o ar para dentro do buraco na marca de lápis, fazendo com que o CAM a cair para longe do reservatório na marca de lápis. </li><li> Verifique se o saco de ar passou do final carimbado do ovo para o buraco marcado-lápis usando o candler. Se não tem, use o pino do impulso para fazer dois buracos um pouco mais profunda e, em seguida, voltar a aplicar sucção usando a lâmpada de segurança. </li><li> Acenda as luzes. Segurando o ovo em uma mão, ligar a ferramenta rotativa Dremel com um 15/16 polegadas roda de corte em anexo e fazer dois cortes transversais apenas profundo o suficiente para cortar a casca, mas não é profundo o suficiente para cortar todo o caminho para o deprimido CAM. Fazer os cortes de cerca de 2 cm de comprimento e 1 cm de distância um do outro. Fazer os cortes de largura suficiente de modo que um anel de silicone de 1 cm de diâmetro pode passar atravésmas mais fino do que a largura de fita adesiva padrão. </li><li> Em seguida, faça um corte longitudinal entre e nas extremidades dos dois cortes transversais tocando levemente a roda de corte para o shell. </li><li> Deslize uma pinça estéril sob a peça shell e paralelo ao shell. Pegue o pedaço shell com a pinça e retire toda a peça limpa. </li><li> Coloque fita adesiva sobre a janela recém-inaugurado, certificando-se de dobrar uma ponta sobre si mesmo para facilitar a remoção. </li><li> Coloque os ovos de volta na incubadora na bandeja do ovo (NOT nas prateleiras de ovos rotativas) até o momento da inoculação. </li></ol> 3. Inoculação <ol><li> Mantenha porão mistura de proteína de membrana, como matrigel, em gelo (para evitar a polimerização) e coloque na capa. </li><li> Use EDTA 2 mM, para separar as células (4 - 5 ml por frasco T75) e frascos de cultura de tecido em lugar incubadora durante 5 min. </li><li> Volte a suspender as células isoladas nos meios de comunicação e contar com um hemocitômetro e azul de tripano. Use entre 500.000 to 2.000.000 de células para enxertia sobre o CAM, dependendo da linha celular. Otimizar o número adequado de experimentos preliminares. </li><li> Medida do volume necessário para o número de células apropriado e centrifugar a 500 xg durante 5 min. </li><li> Descartar o sobrenadante e ressuspender em seguida sedimento celular em 40 μ l de PBS ++ e 20 μ l de mistura de membrana basal. NOTA: Neste ponto, as células podem ser tratadas com fármacos ou outros agentes de teste quanto necessário para as experiências por ressuspensão em suspensão a mistura membrana celular / porão. Alternativamente, os agentes podem ser pipetado para a suspensão de células de membrana mistura / cave após o enxerto. </li><li> Recuperar rack de ovo com os ovos da incubadora e lugar na capela de fluxo laminar. </li><li> Retire a fita da janela, recuperar um anel de silicone 1 mm do tampa de um frasco criogênico usando uma pinça esterilizados, e soltar o anel pela janela. </li><li> Pipetar a solução de células 60 μ l de PBS ++ e membrana basal MIXTURe directamente para o centro do anel de silicone que descansa sobre a CAM. </li><li> Re-tape a janela e mover prateleira dos ovos de volta à incubadora. Neste ponto, tenha cuidado ao mover os ovos como o anel e a suspensão de células pode facilmente se tornar deslocados. </li><li> Permitir que os tumores crescer durante 5 a 7 dias. </li></ol> 4. Colhendo Tumores <ol><li> Coloque sob-pad na capa. Preparar 35 x 10 mm pratos e recipientes de paraformaldeído, conforme necessário para a histologia. Colocar 1 mL de PBS ++ em cada placa de 35 mm x 10. </li><li> Coloque saco de risco biológico na capa. Pegue uma tesoura de dissecção estéril e introduza a ponta para dentro do ovo no buraco perto do final carimbado. </li><li> Corte em torno de todo o ovo longitudinalmente. NOTA: o CAM eo tumor com o anel deve ficar para a metade superior do reservatório com a janela. </li><li> Com uma tesoura de dissecção e uma pinça, dissecar o tumor longe do CAM e coloque tumor na placa de 35 mm x 10. Medir e pesar tumores. Tumores fixas em paraformaldehyde parafina e, em seguida, incorporar-los para uso em histologia e imunohistoquímica experimentos. </li></ol> 5. (Opcional): Tumor de dissociação de células <ol><li> Use tesouras de dissecação para picar o tumor na placa de 35 mm x 10. Pour tumor fragmentado em um tubo de centrífuga de 15 ml e remover o máximo de PBS ++ quanto possível através de pipetagem. </li><li> Adicionar 2 ml de colagenase (1 mg / ml em PBS ++), ao longo do tubo ligar várias vezes, e incubar a 37 ° tubo C durante pelo menos 30 minutos (até várias horas). </li><li> Adicionar 5 ml de mídia para cada tubo e ressuspender completamente pipetando cima e para baixo 20 - 40x. Pipetagem minuciosa é fundamental para a dissociação de células completa. </li><li> Permitir a sedimentação, e transferir o sobrenadante para um novo tubo. Centrifugar a 500 xg durante 5 min. Remover o sobrenadante, deixando para trás pelete de células dissociadas. NOTA: neste momento, os sedimentos celulares podem ser usadas para várias experiências (citometria de fluxo, a lise para uso na transferência de Western, o isolamento de ARN, etc.). A contagem total de células tumorais também pode ser obtido usando um hemocitómetro e coloração com azul de tripano. Note-se que, células ovais menores são células de galinha do CAM e não devem ser contadas como células tumorais. Deve haver um número mínimo de contaminação de células não tumorais (CAM) Se o tumor é dissecada cuidadosamente a partir da CAM. </li></ol>

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A.S.&#160;receives&#160;funding from the Advaxis Pharmaceutical company to support an investigator-initiated clinical trial.&#160;

Vários passos principais neste protocolo mais provavelmente representam a sobrevivência embrionária melhorada bem como uma maior fiabilidade do crescimento do tumor. Soltando o CAM para longe do reservatório através da aplicação de sucção para o saco de ar é menos invasiva do que os outros métodos existentes (utilizando uma agulha para remover a albumina do ovo, dissecção cega, etc.). Usando um alfinete estéril para criar os dois pequenos furos necessários para este método é a parte mais desafiadora do ...

O carcinoma hepatocelular (HCC) é a 3ª maior causa de mortalidade por câncer no mundo 1. Atualmente, apenas 30% dos pacientes com CHC são elegíveis para os tratamentos cirúrgicos potencialmente curativas 2, e quimioterapia sistêmica não é eficaz 3. Portanto, existe uma necessidade premente para clínica não satisfeita terapias HCC novos, e o desenvolvimento de sistemas modelo adequados para o teste eficiente de novos agentes. O corioalantï¿½ca membrana (CAM) ensaio pintainho fornece um método reprodutível, de custo-eficaz, rápido e de médio rendimento de testar potenciais fármacos anti-tumorais in vivo. O ensaio CAM tem sido amplamente utilizado para estudar angiogénese 4. Também foi desenvolvido com sucesso num modelo de xenoenxerto de tumor de cancros, incluindo o glioblastoma 5, 6 cancro pancreático, melanoma 7-9, 10-11 e osteossarcoma. Ambos in ovo 12 e ex ovo13 técnicas têm sido utilizadas na literatura, com detalhes que variam de protocolo para protocolo. Um grande desafio para o modelo CAM xenotransplante é a incidência relativamente elevada de morte embrionária após a manipulação do ovo, com taxas de mortalidade de embriões de pintainho publicados que variam 25-50 por cento 11-14. Neste artigo, descrevemos o desenvolvimento de um modelo in ovo de xenotransplante de HCC que produz de forma confiável crescimento de tridimensionais, tumores vascularizados que histologicamente se assemelham HCC indiferenciado. Temos adaptado um protocolo inicialmente descrito por Ossowski et ai. 14 e ter atingido pintainho embrionário taxas de sobrevivência de até 93% com o enxerto do tumor extremamente alta.

<table border="0" cellpadding="0" cellspacing="0" width="1094">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Name of Reagent/ Equipment</td>
			<td style="width:108px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:467px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Premium incubated eggs</td>
			<td style="width:108px;">Charles River</td>
			<td style="width:119px;">N/A</td>
			<td>http://www.criver.com/files/pdfs/avian/av_c_spf_egg_price_list.aspx</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:401px;">Egg incubators</td>
			<td style="width:108px;">GQF</td>
			<td style="width:119px;">Hova-Bator 2362N</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:401px;">Rotating egg trays</td>
			<td style="width:108px;">GQF</td>
			<td style="width:119px;">1611 Automatic Egg Turner</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:401px;">Egg candler</td>
			<td style="width:108px;">Lyon</td>
			<td style="width:119px;">Hi-Power 950-070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Dremel 100 rotary tool with 15/16 cut-off wheel</td>
			<td style="width:108px;">Dremel</td>
			<td style="width:119px;">100-N/7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Sterile forceps, push pin, dissection scissors, Scotch tape</td>
			<td style="width:108px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Matrigel</td>
			<td style="width:108px;">BD Biosciences</td>
			<td style="width:119px;">356234</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Cryogenic vials, external thread with silicone washer</td>
			<td style="width:108px;">Corning</td>
			<td style="width:119px;">430659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Collagenase from Clostridium histolyticum</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:119px;">C9891</td>
			<td></td>
		</tr>
	</tbody>
</table>

the in ovo chick chorioallantoic membrane (cam) assay as an efficient xenograft model of hepatocellular carcinoma

A membrana corioalantï¿½ca pinto (CAM) começa a se desenvolver no dia 7 após a fertilização e amadurece a dia 12. O CAM é naturalmente imunodeficientes e altamente vascularizado, tornando-se um sistema ideal para a implantação do tumor. Além disso, a CAM contém proteínas da matriz extracelular, tais como fibronectina, laminina, colagénio, integrina alfa (V) beta3, e MMP-2, tornando-se um modelo atractivo para estudar a invasão tumoral e metástases. Os cientistas têm tomado longo vantagem da fisiologia da CAM usando-o como um modelo de angiogénese. Mais recentemente, o ensaio CAM foi modificado para funcionar como um sistema modelo in vivo de xenotransplante para vários tipos de câncer que preenche a lacuna entre o trabalho de base in vitro e modelos mais complexos de cancro animal. O ensaio CAM permite o estudo do crescimento do tumor, as terapias anti-tumorais, e vias moleculares pró-tumorais num sistema que é biologicamente importante tanto em termos de custos e tempo eficaz. Aqui, descrevemos o desenvolvimento da CAM xenograft modelo de carcinoma hepatocelular (HCC), com taxas de sobrevivência de embriões de até 93% e tumor confiável tomar levando a um crescimento de tridimensionais, tumores vascularizados.

Imagens representativas dos passos chave no protocolo são mostrados aqui. A Figura 1A demonstra o uso do candler para visualizar o embrião em desenvolvimento, o saco de ar, e a vasculatura da CAM. A Figura 1B-1C mostram o processo de deixar cair a CAM, fazendo os dois orifícios e, em seguida, a aplicação de pressão negativa usando a lâmpada de segurança, e a Figura 1D mostra uma removida com sucesso CAM com uma bolha de ar grande sob o buraco ma...

Watch this Scientific Journal Video about The In Ovo Chick Chorioallantoic Membrane CAM Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma at JoVE.com

The In Ovo Chick Chorioallantoic Membrane CAM Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

The chick chorioallantoic membrane (CAM) is immunodeficient and highly vascularized, making it a natural in vivo model of tumor growth and angiogenesis. In this protocol, we describe a reliable method of growing three-dimensional, vascularized hepatocellular carcinoma (HCC) tumors using the CAM assay.

o<em&gt; In Ovo</em&gt; Chick Corioalantóide membrana (CAM) como um Ensaio Xenoenxerto modelo eficiente de carcinoma hepatocelular

The chick chorioallantoic membrane (CAM) begins to develop by day 7 after fertilization and matures by day 12. The CAM is naturally immunodeficient and ...

the-ovo-chick-chorioallantoic-membrane-cam-assay-as-an-efficient

Research

JoVE Journal

Medicine

The In Ovo Chick Chorioallantoic Membrane (CAM) Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

34.9K visualizações.  Icahn School of Medicine at Mount Sinai. The chick chorioallantoic membrane (CAM) is immunodeficient and highly vascularized, making it a natural in vivo model of tumor growth and angiogenesis. In this protocol, we describe a reliable method of growing three-dimensional, vascularized hepatocellular carcinoma (HCC) tumors using the CAM assay.

Vídeo: The In Ovo Chick Chorioallantoic Membrane CAM Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Multi-fotão imagem de invasão das células tumorais em um modelo de camundongo ortotópico de carcinoma epidermóide oral

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Medicina

An Orthotopic Mouse Model of Anaplastic Thyroid Carcinoma

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer cells into immunodeficient mice. Subcutaneous xenograft models have been valuable for preclinical screening and evaluation of new therapeutic treatments. There are a number of advantages to using a subcutaneous model; 1) rapid, 2) reproducible, and 3) tumor establishment, growth, and response to therapeutic agents may be monitored by visual inspection. However, substantial evidence has shed light on the short-comings of subcutaneous xenograft models1-3. For instance, medicinal treatments demonstrating curative properties in subcutaneous xenograft models often have no notable impact on the human disease. The microenvironment of the site of xenographic transplantation or injection lies at the heart of this dissimilarity.
Orthotopic tumor xenograft models provide a more biologically relevant context in which to study the disease. The advantages of implanting diseased cells or tissue into their anatomical origin equivalent within a host animal includes a suitable site for tumor-host interactions, development of disease-related metastases and the ability to examine site-specific influence on investigational therapeutic remedies. Therefore, orthotopic xenograft models harbor far more clinical value because they closely reproduce human disease. For these reasons, a number of groups have taken advantage of an orthotopic thyroid cancer model as a research tool4-7.
Here, we describe an approach that establishes an orthotopic model for the study of anaplastic thyroid carcinoma (ATC), which is highly invasive, resists treatment, and is virtually fatal in all diagnosed patients. Cultured ATC cells are prepared as a dissociated cellular suspension in a solution containing a basement membrane matrix. A small volume is slowly injected into the right thyroid gland. Overall appearance and health of the mice are monitored to ensure minimal post-operative complications and to gauge pathological penetrance of the cancer. Mice are sacrificed at 4 weeks, and tissue is collected for histological analysis. Animals may be taken at later time-points to examine more advance progression of the disease. Production of this orthotopic mouse model establishes a platform that accomplishes two objectives: 1) further our understanding of ATC pathology, and 2) screen current and future therapeutic agents for efficacy in combating ATC.

Generation of an orthotopic mouse model of anaplastic thyroid carcinoma is described here. This technique employs surgical placement of human anaplastic thyroid cancer cells into the thyroid of immunodeficient mice, thus creating a more clinically relevant setting to study disease progression as well as screen innovative therapeutic interventions.

Um modelo de camundongo ortotópico de Carcinoma anaplásico da tiróide

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer ...

The In ovo CAM-assay as a Xenograft Model for Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions.
This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products.

The In ovo CAM-assay as a Xenograft Model for Sarcoma

The in ovo chorioallantoic membrane (CAM) is grafted with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior.

O<em&gt; Em ovo</em&gt; CAM-ensaio como um Xenograftmodel para o Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for ...

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

In vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate the human disease provide a valuable platform for research into disease pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.

Human tumor xenografts in immunodeficient mice are valuable tools to study cancer biology. Specific protocols to generate subcutaneous and intrahepatic xenografts from human hepatocellular carcinoma cells or tumor fragments are described. Liver regeneration induced by partial hepatectomy in recipient mice is presented as a strategy to facilitate intrahepatic engraftment.

Geração de subcutânea e intra-hepática Humanos carcinoma hepatocelular Xenoenxertos em Immunodeficient Ratos

In  vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate  the human disease provide a valuable platform for research into disease  ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Excitotóxico Estimulação do Microslices cérebro como um<em&gt; In vitro</em&gt; Modelo de AVC

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Confocal Time Lapse Imaging as an Efficient Method for the Cytocompatibility Evaluation of Dental Composites

It is generally accepted that in vitro cell material interaction is a useful criterion in the evaluation of dental material biocompatibility. The objective of this study was to use 3D CLSM time lapse confocal imaging to assess the in vitro biocompatibility of dental composites. This method provides an accurate and sensitive indication of viable cell rate in contact with dental composite extracts. The ELS extra low shrinkage, a dental composite used for direct restoration, has been taken as example. In vitro assessment was performed on cultured primary human gingival fibroblast cells using Live/Dead staining. Images were obtained with the FV10i confocal biological inverted system and analyzed with the FV10-ASW 3.1 Software. Image analysis showed a very slight cytotoxicity in the presence of the tested composite after 5 hours of time lapse. A slight decrease of cell viability was shown in contact with the tested composite extracts compared to control cells. The findings highlighted the use of 3D CLSM time lapse imaging as a sensitive method to qualitatively and quantitatively evaluate the biocompatibility behavior of dental composites.

The most studied aspect of the biocompatibility of dental composites is their cytotoxicity 3D CLSM time lapse imaging combined with fluorescent signal quantification allows sensitive evaluation of the cell fate over time. Utilizing this method could be an efficient tool to assess the cytocompatibility of dental composites.

Confocal Time Lapse de imagem como um método eficiente para a Avaliação citocompatibilidade of Dental Composites

It is generally accepted that in vitro cell material interaction is a useful criterion in the evaluation of dental material biocompatibility. The ...

A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of cancer metastasis are still unclear. Animal models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of metastatic cancers. Here, an in-depth description of a &#8220;patient-like&#8221; orthotopic syngeneic mouse model for exploring the mechanisms of metastasis of solid organ tumors is provided. The survival surgical implantation of BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c is described. The success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of solid organ cancer metastasis.

A &quot;Patient-Like&quot; Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The metastatic spread of cancer is the major cause of cancer-related deaths. We provide an in-depth description of our survival surgery methodology for establishing a &#8220;patient-like&#8221; orthotopic syngeneic mouse model system for studying the mechanisms of metastasis in solid organ tumors.

A &quot;Patient-Like&quot; Orthotopic Singênico Rato Modelo de carcinoma hepatocelular Metástase

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of ...

Designing CAD/CAM Surgical Guides for Maxillary Reconstruction Using an In-house Approach

Computer-aided design/computer-assisted manufacturing (CAD/CAM) is now being evaluated as a preparative technique for maxillofacial surgery. Because this technique is expensive and available in only limited areas of the world, we developed a novel CAD/CAM surgical guide using an in-house approach. By using the CAD software, the maxillary resection area and cutting planes and the fibular cutting planes and angles are determined. Once the resection area is decided, the necessary faces are extracted using a Boolean modifier. These superficial faces are united to fit the surface of the bones and thickened to stabilize the solids. Not only the cutting guides for the fibula and maxilla but also the location arrangement of the transferred bone segments is defined by thickening the superficial faces. The CAD design is recorded as .stl files and three-dimensionally (3-D) printed as actual surgical guides. To check the accuracy of the guides, model surgery using 3-D-printed facial and fibular models is performed. These methods may be used to assist surgeons where commercial guides are not available.

Methods for designing a computer-aided design/computer-aided manufacturing (CAD/CAM) surgical guide are shown. Cutting planes are separated, united, and thickened to easily visualize the necessary bone transfer. These designs can be three-dimensional printed and checked for accuracy.

Concepção CAD/CAM guias cirúrgicos para reconstrução maxilar usando uma abordagem In-house

Computer-aided design/computer-assisted manufacturing (CAD/CAM) is now being evaluated as a preparative technique for maxillofacial surgery. Because this ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Medição de contratilidade isométrica da artéria mesentérica Mouse usando fio miografia

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Predicting Treatment Response to Image-Guided Therapies Using Machine Learning: An Example for Trans-Arterial Treatment of Hepatocellular Carcinoma

Intra-arterial therapies are the standard of care for patients with hepatocellular carcinoma who cannot undergo surgical resection. The objective of this study was to develop a method to predict response to intra-arterial treatment prior to intervention.
The method provides a general framework for predicting outcomes prior to intra-arterial therapy. It involves pooling clinical, demographic and imaging data across a cohort of patients and using these data to train a machine learning model. The trained model is applied to new patients in order to predict their likelihood of response to intra-arterial therapy.
The method entails the acquisition and parsing of clinical, demographic and imaging data from N patients who have already undergone trans-arterial therapies. These data are parsed into discrete features (age, sex, cirrhosis, degree of tumor enhancement, etc.) and binarized into true/false values (e.g., age over 60, male gender, tumor enhancement beyond a set threshold, etc.). Low-variance features and features with low univariate associations with the outcome are removed. Each treated patient is labeled according to whether they responded or did not respond to treatment. Each training patient is thus represented by a set of binary features and an outcome label. Machine learning models are trained using N - 1 patients with testing on the left-out patient. This process is repeated for each of the N patients. The N models are averaged to arrive at a final model.
The technique is extensible and enables inclusion of additional features in the future. It is also a generalizable process that may be applied to clinical research questions outside of interventional radiology. The main limitation is the need to derive features manually from each patient. A popular modern form of machine learning called deep learning does not suffer from this limitation, but requires larger datasets.

Intra-arterial therapies are the standard of care for patients with hepatocellular carcinoma who cannot undergo surgical resection. A method for predicting response to these therapies is proposed. The technique uses pre-procedural clinical, demographic, and imaging information to train machine learning models capable of predicting response prior to treatment.

Prevendo a resposta do tratamento para terapias guiada por imagem utilizando aprendizado de máquina: um exemplo para o Trans-Arterial tratamento do Carcinoma hepatocelular

Intra-arterial therapies are the standard of care for patients with hepatocellular carcinoma who cannot undergo surgical resection. The objective of this ...