Este trabalho foi apoiado pela Associazione Italiana per la Ricerca sul Cancro, italiano Ministério da Universidade e da Pesquisa, projetos Prin e Progetti di Ricerca di Ateneo, conceder N ° CPDA137871, Fondazione Cariplo, conceder N ° 2011-0485 para MDB, e por Finanziamento Giovani studiosi, da Universidade de Pádua, para Gaia Codolo.

Todos os procedimentos para lidar com os animais foram aprovados pelo Ministério italiano da Saúde (204/2011-B DM). 1. Os animais <ol><li> Crescer camundongos fêmeas C57BL6 / J 8 semanas de idade em gaiolas ventiladas individualmente com filtros microisolation. Nota: As fêmeas são preferidos devido à conformação anatómica do seu aparelho de uro-genital externa torna o cateterismo mais fácil do que nos machos. </li></ol> 2. Cultura celular <ol><li> Manter a linha celular de carcinoma urotelial rato MB49 em RPMI 1640 meio suplementado com 10% de soro bovino fetal inactivado pelo calor e 50 ng / ml de gentamicina, a 37 ° C em 5% humidificada de CO 2. </li><li> Cultivar células MB49 em frascos T-75 a 80% de confluência. Tripsinizar e ressuspender em 0,5 × 10 5-0,5 x 10 6 células por 150 ul de PBS antes da implantação em ratos. </li></ol> 3. Intravesical tumor Implant <ol><li> Anestesiar ratos, utilizando uma mistura de anestésicos e o Zoletil xylor (33 mg / kg e 20 mg / kg, respectivamente), injectada intraperitonealmente. </li><li> Coloque os animais em decúbito dorsal e corrigir as patas traseiras para a almofada com fita adesiva. Patas têm de ser suficientemente separadas para processar o meato uretral bem visíveis e facilmente acessível para a inserção do cateter. </li><li> Nota: Para a cateterismo, um cateter venoso pediátrica 24 G é adequado para ratinhos 8 a 10 semanas de idade. A escolha do cateter é muito importante a fim de evitar a fuga de líquido entre a parede da uretra e do cateter. Cateteres furo maior deve ser utilizado para ratinhos maiores ou mais velhos. </li><li> Usando uma pequena pinça, beliscar uma borda da parte externa da uretra e torcê-lo muito suavemente em direção ao &quot;fim cabeça&quot; dos ratinhos. Esta ação expõe o meato uretral. </li><li> Retirar a agulha do cateter interno e inserir este último na uretra com um ângulo de 45 °, orientada ligeiramenteno sentido da &quot;extremidade posterior&quot; dos ratinhos. Não force a entrada do cateter; em caso de resistência, basta girar o cateter muito suavemente, até que desliza para dentro da uretra. Quando cerca de 0,5-0,8 cm do cateter na uretra é, colocar cuidadosamente o cateter paralelo para a cauda dos ratos e inseri-lo completamente. </li><li> Usando uma seringa de 1 ml, a agulha de que tem de ser inserido no cateter, lava-se a partir de urina residual da bexiga através da injecção de 200-300 ul de PBS estéril e aspirar o líquido a partir da bexiga. </li><li> Nota: Ao injetar PBS, a bexiga vai encher-se e um inchaço será visível entre as patas traseiras dos ratos; isto confirma que o cateterismo é correto. A maioria dos cateteres têm um volume morto que tem de ser considerada quando se calcula o volume de injecção. </li><li> Usando uma seringa esterilizada de 1 ml, injectar 200 ul de 0,1 N de HCl. Preencha a bexiga a fim de queimar toda a sua parede. Após 10 segundos, retire todo o ácido e imediatamente neutralize por injecção de 200 uL de NaOH 0,1 N, com uma seringa estéril. No caso de injecção de maior número de células (0,5 x 10 6), o passo de acidificação não é necessária. </li><li> Aspire todo o líquido residual e imediatamente lavar a cavidade com 300 ï¿½ de PBS estéril. </li><li> Esvaziar a bexiga por aspiração, e injectar 150 ul de PBS contendo células MB49 (intervalo de 0,5 x 10 5-0,5 x 10 6) no interior da bexiga. Deixar a seringa montada no cateter a fim de evitar a fuga de células. É melhor para garantir a seringa, fixando-lo à almofada com fita adesiva. </li><li> Após 1 hora, extrair suavemente o cateter da uretra, e colocar os murganhos na gaiola, sob uma lâmpada incandescente até despertaram: este ocorre geralmente entre 1 e 2 horas após o fim do tratamento. </li></ol> 4. Entrega de Intravesical HP-NAP <ol><li> Nota: pelo menos 3 dias após a instilação de células, é possível começar atratamento. </li><li> Cateterize como descrito nas etapas 3.1 a 3.4. Nesta fase, não é necessário para queimar a parede da bexiga com HCl. </li><li> Usando uma seringa de 1 ml, lava-se a partir de urina residual da bexiga através da injecção de 200-300 ul de PBS estéril e aspirar o líquido a partir da bexiga. </li><li> Injectar 50 ug de HP-NAP por 150 ul de PBS para dentro da bexiga. Deixar a seringa ligado ao cateter para evitar fugas da solução de proteína. Fixe a seringa, fixando-lo à almofada com fita adesiva. </li><li> Após 1 hora, extrair suavemente o cateter da uretra e colocar os murganhos na gaiola, sob uma lâmpada incandescente, até que acordado. </li></ol> 5. tumor explantes e da análise histológica <ol><li> No final do tratamento, sacrificar os animais, colocá-los em posições supina e corrigir as patas traseiras para a almofada com fita adesiva. </li><li> Usando um bisturi cirúrgico, fazer uma incisão vertical sobre a 1,52 centímetros de comprimento através da pele e doperitônio, começando logo acima genitália externa para o &quot;fim cabeça&quot; dos ratinhos. Para minimizar o risco de danificar a bexiga, preste especial atenção para a profundidade do corte. </li><li> A bexiga será posicionado no centro-esquerda do corte e do tumor será visível como uma massa rosa / violeta escuro. Após a sua excisão, colocar toda a bexiga numa gaiola histológica e fixar em 10% de formalina tamponada antes de ser embutido em parafina. </li><li> Para análise histológica e histoquimica imune, preparar secções em série da bexiga (46 uM de espessura) usando um micrótomo padrão. </li><li> Corar as secções com hematoxilina / eosina e prosseguir com o cálculo do tamanho do tumor (D xd 2) e determinar a porcentagem de necrose por área transversal usando um computador assistida analisador de imagem. </li><li> Para a contagem navio, proceder por coloração das secções para o marcador endotelial CD31 / PECAM, usando uma técnica de imunoperoxidase padrão. Digitalizar as seções umt baixa potência (4 x) para identificar a área de maior densidade vascular. Dentro desta região, contar microvasos individuais em cinco campos aleatórios separados em alta potência (20 ×). </li></ol>

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immune+modulating+activity+of+the+Helicobacter+pylori+HP-NAP:+Friend+or+foe.">The immune modulating activity of the Helicobacter pylori HP-NAP: Friend or foe.</a> Toxicon. 56, 1186-1192 (2010).</li><li>Codolo, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neutrophil-activating+protein+of+Helicobacter+pylori+down-modulates+Th2+inflammation+in+ovalbumin-induced+allergic+asthma.">The neutrophil-activating protein of Helicobacter pylori down-modulates Th2 inflammation in ovalbumin-induced allergic asthma.</a> Cell Microbiol. 10, 2355-2363 (2008).</li><li>Gunther, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+syngeneic+orthotopic+murine+bladder+cancer+(MB49).">Optimizing syngeneic orthotopic murine bladder cancer (MB49).</a> Cancer Res. 59, 2834-2837 (1999).</li><li>Codolo, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HP-NAP+inhibits+the+growth+of+bladder+cancer+in+mice+by+activating+a+cytotoxic+Th1+response.">HP-NAP inhibits the growth of bladder cancer in mice by activating a cytotoxic Th1 response.</a> Cancer Immunol Immunother. 61, 31-40 (2012).</li><li>Chan, E., Patel, A., Heston, W., Larchian, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+orthotopic+models+for+bladder+cancer+research.">Mouse orthotopic models for bladder cancer research.</a> BJU Int. 104, 1286-1291 (2009).</li><li>Kubota, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastatic+models+of+human+cancer+xenografted+in+the+nude+mouse:+the+importance+of+orthotopic+transplantation.">Metastatic models of human cancer xenografted in the nude mouse: the importance of orthotopic transplantation.</a> J Cell Biochem. 56, 4-8 (1994).</li><li>Loi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+the+orthotopic+model+to+validate+antivascular+therapies+for+cancer.">The use of the orthotopic model to validate antivascular therapies for cancer.</a> Int J Dev Biol. 55, 547-555 (2011).</li><li>Miyazaki, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preconditioning+methods+influence+tumor+property+in+an+orthotopic+bladder+urothelial+carcinoma+rat+model.">Preconditioning methods influence tumor property in an orthotopic bladder urothelial carcinoma rat model.</a> Mol Clin Oncol. 2, 65-70 (2014).</li><li>Horiguchi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+orthotopic+mouse+superficial+bladder+tumor+model+for+studies+on+intravesical+treatments.">Establishment of orthotopic mouse superficial bladder tumor model for studies on intravesical treatments.</a> Hum Cell. 21, 57-63 (2008).</li></ol>

Os autores declaram que não têm interesses financeiros concorrentes.

A maioria dos avanços na terapia do cancro requerem testes em modelos animais antes de começar testes clínicos. A possibilidade de estudar a biologia do tumor in vivo, aproveitando-se de modelos animais, representa uma ferramenta crucial para os pesquisadores que investigam patogênese do câncer, permitindo a avaliação de diferentes abordagens terapêuticas. Modelos ortotópicos sendo o padrão-ouro 14-15, tanto por causa da enorme quantidade de linhas celulares disponíveis para configurar um ...

O câncer de bexiga é um dos cânceres mais comuns do trato urogenital, com cerca de 75.000 novos casos por ano nos EUA 1. As altas taxas de recorrência ao longo da vida exigem acompanhamento, o que torna o câncer de bexiga um dos cancros mais caras para tratar. O tratamento padrão ouro para o alto grau NMIBC é a ressecção trans-uretral, seguida de imunoterapia intravesical com BCG. Embora o mecanismo exacto da actividade anti-tumoral de BCG continua a ser totalmente elucidado, admite-se que a activação de uma resposta imune mediada por células enriquecida em células T auxiliares (Th) e um T citotóxicas (Tc) um células é crucial para o sucesso da terapia de dois. Apesar do fato que a BCG continua a ser o tratamento de escolha para NMIBC, uma elevada proporção de pacientes não responde à terapia; além disso, pode provocar um número de efeitos secundários: cerca de 70% dos tumores tratados recorrer após algum tempo e ~ 15% para a forma de progresso músculo-invasiva dadoença. Outros efeitos secundários associados com o tratamento com BCG incluem disúria, cistite e infecção renal 3-6. O desenvolvimento de novas estratégias terapêuticas deve levar em consideração a utilização de modelos pré-clínicos, seguindo in vitro de avaliação inicial; isto é particularmente relevante em tumores cuja microambiente pode influenciar significativamente o seu desenvolvimento e capacidade de resposta ao tratamento. Ao longo da última década, têm abordado vários aspectos da actividade de modulação imunitária de HP-PNA, uma proteína produzida pela bactéria Helicobacter pylori, inicialmente identificados como capazes de promover a adesão endotelial de células polimorfonucleares (PMN) 7. Estruturalmente, HP-NAP pertence à proteína-protegendo o DNA sob condições de privação (DPS) da família 8 e é composto por 12 subunidades idênticas dispostas em um shell dodecameric. Nós demonstramos que a HP-NAP éum receptor de tipo Toll (TLR) 2 agonista, com uma forte actividade de imuno-regulação responsáveis ​​por dirigir a diferenciação de linfócitos T no sentido do fenótipo Th1, tanto in vitro como in vivo 9-10. Em virtude desta actividade, HP-NAP é capaz de redirecionar a resposta imune Th2 na resposta mais benéfico Th1 em um modelo murino de asma alérgica 11. Para avaliar o potencial anti-tumoral Th1-dependente de HP-NAP, aproveitamos um mouse modelo de cancro da bexiga desenvolvido há vários anos por O&#39;Donnel e colegas 12 para avaliar o impacto da vacina BCG. Com este protocolo, demonstramos que a HP-NAP tem um potencial anti-tumoral forte contra o cancro da bexiga e que a eficácia da administração de HP-NAP é paralela à acumulação significativa, dentro de nós tumorais e linfonodos regionais, de ambos os linfócitos Th1 e Tc1 produtoras de Interferon ( IFN) -γ 13 </sup&gt;. Os tumores isolados de camundongos tratados com HP-NAP mostrou mais de necrose e menor vascularização do que a contrapartida não tratada. O presente relatório proporciona um protocolo stepbystep, detalhando a preparação dos animais e dos respectivos cateterismo uretral, a queima química necessária para a fixação de células à mucosa da bexiga, e a injecção das células tumorais. Nós também descrevem a administração tópica de HP-PNA que pode ser considerada como um protótipo de quaisquer agentes terapêuticos desenvolvidos para o tratamento de cancro da bexiga. A prova obtida através da comparação de tumores isolados de controle e-tratados com NAP HP animais enfatiza não só o fato de que a HP-NAP pode ser um bom candidato para imunoterapia do cancro de bexiga, mas também a eficácia geral da montagem experimental.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Materials</td>
		</tr>
		<tr>
			<td>C57BL/6J female mice</td>
			<td>Harlan Italy (Udine, Italy)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MB49 Cells</td>
			<td></td>
			<td></td>
			<td>Obtained from Prof. O&#39;Donnel, University of Iowa Carver College of Medicine, Iowa, USA</td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Sigma-Aldrich (St. Louis, Missouri, USA)</td>
			<td>R8758</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Sigma-Aldrich</td>
			<td>F7524</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma-Aldrich</td>
			<td>D1408</td>
			<td>10X, to be diluted in apyrogenic water</td>
		</tr>
		<tr>
			<td>Flask</td>
			<td>Becton Dickinson (Franklin Lakes, New Jersey, USA)</td>
			<td>353135</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe 1ml</td>
			<td>Becton Dickinson</td>
			<td>301358</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Life Technologies (Waltham, Massachusetts, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamycin</td>
			<td>Life Technologies</td>
			<td>15710-049</td>
			<td></td>
		</tr>
		<tr>
			<td>Xilor</td>
			<td>Bio 98 s.r.l. (Milano, Italy)</td>
			<td></td>
			<td>2% Xylazin</td>
		</tr>
		<tr>
			<td>Zoletil</td>
			<td>Virbac (Carros, France)</td>
			<td>359713301992</td>
			<td>5% Zolazepam + 5% Tiletamine</td>
		</tr>
		<tr>
			<td>24G Catheter</td>
			<td>Terumo (Rome, Italy)</td>
			<td>SR+DM2419PX</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Carlo Erba Reagents (Milano, Italy)</td>
			<td>403871</td>
			<td>Liquid</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>JT Baker (Center Valley, Pennsylvania, USA)</td>
			<td>10095011</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Surgical Scalpel</td>
			<td>Albion Surgical Limited (Sheffield, England)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Leica Microsystem ( Wetzlar, Germany)</td>
			<td>RM2235</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Slides</td>
			<td>VWR International (Radnor, Pennsylvania, USA)</td>
			<td>631-0108</td>
			<td></td>
		</tr>
		<tr>
			<td>Image Analyzer</td>
			<td>Zeiss (Jena, Germany)</td>
			<td>Cyres System</td>
			<td></td>
		</tr>
	</tbody>
</table>
Materials C57BL/6J female mice Harlan Italy (Udine, Italy) MB49 Cells Obtained from Prof. O&#39;Donnel, University of Iowa Carver College of Medicine, Iowa, USA RPMI Sigma-Aldrich (St. Louis, Missouri, USA) R8758 FBS Sigma-Aldrich F7524 PBS Sigma-Aldrich D1408 10X, to be diluted in apyrogenic water Flask Becton Dickinson (Franklin Lakes, New Jersey, USA) 353135 Syringe 1ml Becton Dickinson 301358 Trypsin Life Technologies (Waltham, Massachusetts, USA) Gentamycin Life Technologies 15710-049 Xilor Bio 98 s.r.l. (Milano, Italy) 2% Xylazin Zoletil Virbac (Carros, France) 359713301992 5% Zolazepam + 5% Tiletamine 24G Catheter Terumo (Rome, Italy) SR+DM2419PX HCl Carlo Erba Reagents (Milano, Italy) 403871 Liquid NaOH JT Baker (Center Valley, Pennsylvania, USA) 10095011 Powder Equipment Surgical Scalpel Albion Surgical Limited (Sheffield, England) Microtome Leica Microsystem ( Wetzlar, Germany) RM2235 Microscope Slides VWR International (Radnor, Pennsylvania, USA) 631-0108 Image Analyzer Zeiss (Jena, Germany) Cyres System

evaluation of the efficacy of the h. pylori protein hp-nap as a therapeutic tool for treatment of bladder cancer in an orthotopic murine model

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold standard treatment for the high-grade non-muscle invasive bladder cancer (NMIBC). However, since the treatment-related side effects are relevant, newer biological response modifiers with a better benefit/side effects ratio are needed.
The tumour microenvironment can influence both tumour development and therapy efficacy. In order to obtain a good model, it is desirable to implant tumour cells in the organ from which the cancer originates.
In this protocol, we describe a method for establishing a tumour in the bladder cavity of female mice and subsequent delivery of therapeutic agents; the latter are exemplified by our use of Helicobacter pylori neutrophil activating protein (HP-NAP). A preliminary chemical burn of the mucosa, followed by the injection of mouse urothelial carcinoma cell line MB49 via urethral catheterization, enables the cells to attach to the bladder mucosa. After a period, required to allow an initial proliferation of the cells, mice are treated with HP-NAP, administrated again via catheterization. The anti-tumour activity of HP-NAP is evaluated comparing the tumour volume, the extent of necrosis and the degree of vascularization between vehicle- and HP-NAP-treated animals.

A Figura 1 mostra a técnica de cateterização; o mouse é cateterizada e instilada com 0,5 × 10 6 células MB49. O tratamento com HP-NAP foi iniciado 3 dias após a injecção de células tumorais, para lhes permitir fixar à parede da bexiga e proliferar. Todos os animais que pertencem ao grupo de controlo desenvolver o tumor; alguns deles podem morrer, antes do final do período de 13 dias, devido à oclusão da uretra. Após a primeira injecção com HP-PNA,...

Watch this Scientific Journal Video about Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model at JoVE.com

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

Here the method to establish a syngeneic mouse model of orthotopic bladder tumour to evaluate the anti-tumour efficacy of the bacterial protein HP-NAP is described.

Avaliação da Eficácia da<em&gt; H. pylori</em&gt; Protein HP-NAP como uma ferramenta terapêutica para o tratamento de cancro da bexiga em um Modelo murino Orthotopic

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold ...

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Research

JoVE Journal

Medicine

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

7.2K visualizações.  University of Padua. Here the method to establish a syngeneic mouse model of orthotopic bladder tumour to evaluate the anti-tumour efficacy of the bacterial protein HP-NAP is described.

Vídeo: Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

An Orthotopic Model of Murine Bladder Cancer

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and subsequent cell adhesion. After their bladders are transurethrally catheterized and drained, animals are again catheterized to permit insertion of a platinum wire into bladders without damaging the urethra or bladder. The catheters are made of Teflon to serve as an insulator for the wire, which will conduct electrical current into the bladder to create a burn injury. An electrocautery unit is used to deliver 2.5W to the exposed end of the wire, burning away extracellular layers and providing attachment sites for carcinoma cells that are delivered in suspension to the bladder through a subsequent catheterization. Cells remain in the bladder for 90 minutes, after which the catheters are removed and the bladders allowed to drain naturally. The development of tumor is monitored via ultrasound. Specific attention is paid to the catheterization technique in the accompanying video.

Bladder tumors are established in female mice in a minimally invasive fashion through catheterization, local cauterization, and subsequent adhesion of carcinoma cells to the burn sites.

Um modelo ortotópico de câncer de bexiga murino

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and ...

Medicina

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 ...

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Biomarcadores em um modelo animal para Revelando correlatos neurais, hematológica e Comportamental do TEPT

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder tumor model. The mouse model is established by urethral catheterization under inhaled general anesthetic. Chemical burn is then introduced to the bladder mucosa using intravesical silver nitrate solution to disrupt the bladder glycosaminoglycan layer and allows cells to attach. Following several washes with sterile water, human bladder cancer KU-7-luc2-GFP cells are instilled through the catheter into the bladder to dwell for 2 hours. Subsequent growth of bladder tumors is confirmed and monitored by in vivo bladder ultrasound and bioluminescent imaging. The tumors are then treated intravesically with saRNA formulated in lipid nanoparticles (LNPs). Tumor growth is monitored with ultrasound and bioluminescence. All steps of this procedure are demonstrated in the accompanying video.

Establishing an orthotopic bladder tumor model to evaluate antitumor effects of intravesically delivered saRNA and monitoring tumor growth by ultrasound and bioluminescent imaging.

Um modelo de tumor de bexiga ortotópico e Avaliação de Tratamento de sarna intravesical

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder ...

An Orthotopic Bladder Cancer Model for Gene Delivery Studies

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are needed. The tumor microenvironment can significantly influence tumor development and therapy response. It is therefore often desirable to grow tumor cells in the organ from which they originated. This protocol describes an orthotopic model of bladder cancer, in which MB49 murine bladder carcinoma cells are instilled into the bladder via catheterization. Successful tumor cell implantation in this model requires disruption of the protective glycosaminoglycan layer, which can be accomplished by physical or chemical means. In our protocol the bladder is treated with trypsin prior to cell instillation. Catheterization of the bladder can also be used to deliver therapeutics once the tumors are established. This protocol describes the delivery of an adenoviral construct that expresses a luciferase reporter gene. While our protocol has been optimized for short-term studies and focuses on gene delivery, the methodology of mouse bladder catheterization has broad applications.

Implantation of cancer cells into the organ of origin can serve as a useful preclinical model to evaluate novel therapies. MB49 bladder carcinoma cells can be grown within the bladder following intravesical instillation. This protocol demonstrates catheterization of the mouse bladder for the purpose of tumor implantation and adenoviral delivery.

Um modelo de cancro da bexiga para Ortotópico Gene Estudos de entrega

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

Um Modelo murino de próstata humana Metástase Ortotópico

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable cell lines are inoculated either by intravesical instillation (model of nonmuscle invasive growth) or intramural injection into the bladder wall (model of invasive growth). Both procedures are complex and highly time-consuming. Additionally, the superficial model has its shortcomings due to the lack of cell lines that are tumorigenic following instillation. Intramural injection, on the other hand, is marred by the invasiveness of the procedure and the associated morbidity for the host mouse.
With these shortcomings in mind, we modified previous methods to develop a minimally invasive approach for creating orthotopic bladder cancer xenografts. Using ultrasound guidance we have successfully performed percutaneous inoculation of the bladder cancer cell lines UM-UC1, UM-UC3 and UM-UC13 into 50 athymic nude. We have been able to demonstrate that this approach is time efficient, precise and safe. With this technique, initially a space is created under the bladder mucosa with PBS, and tumor cells are then injected into this space in a second step. Tumor growth is monitored at regular intervals with bioluminescence imaging and ultrasound. The average tumor volumes increased steadily in in all but one of our 50 mice over the study period.
In our institution, this novel approach, which allows bladder cancer xenograft inoculation in a minimally-invasive, rapid and highly precise way, has replaced the traditional model.

The established technique to inoculate primary invasive orthotopic bladder cancer xenografts requires laparotomy and mobilization of the bladder. This procedure inflicts significant morbidity on the mice, is technically challenging and time-consuming. We therefore developed a high-precision, percutaneous approach utilizing ultrasound guidance.

Minimamente Invasiva Estabelecimento de murino ortotópico bexiga Xenoenxertos

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable ...

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 ...

Pre-clinical Orthotopic Murine Model of Human Prostate Cancer

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information about this disease. The human orthotopic prostate cancer xenograft mouse model provides a useful alternative approach for understanding the specific interactions between genetically and molecularly altered tumor cells, their organ microenvironment, and for evaluation of efficacy of therapeutic regimens. This is a well characterized model designed to study the molecular events of primary tumor development and it recapitulates the early events in the metastatic cascade prior to embolism and entry of tumor cells into the circulation. Thus it allows elucidation of molecular mechanisms underlying the initial phase of metastatic disease. In addition, this model can annotate drug targets of clinical relevance and is a valuable tool to study prostate cancer progression. In this manuscript we describe a detailed procedure to establish a human orthotopic prostate cancer xenograft mouse model.

Prostate cancer is the second most common cause of cancer-related deaths in the United States. An orthotopic cancer model provides a useful approach to understand the biology of prostate cancer and to evaluate the efficacy of therapeutic regimens. This protocol describes detailed steps necessary to establish an orthotopic prostate cancer mouse model.

Pré-clínica Ortotópico Modelo murino de cancro da próstata humano

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information ...

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking (TXL) for Myopia

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy include photochemical cross-linking and tissue cross-linking (TXL) methods. Such methods for inducing mechanical tissue property changes are being employed to the cornea in corneal thinning (mechanically weakened) disorders such as keratoconus as well as the sclera in progressive myopia, where thinning and weakening of the posterior sclera occurs and likely contributes to axial elongation. The primary target proteins for such tissue strengthening are fibrillar collagens which constitute the great majority of dry weight proteins in the cornea and sclera. Fortuitously, fibrillar collagens are the main source of second harmonic generation signals in the tissue extracellular space. Therefore, modifications of the collagen proteins, such as those induced through cross-linking therapies, could potentially be detected and quantitated through the use of second harmonic generation microscopy (SHGM). Monitoring SHGM signals through the use of a laser scanning microscopy system coupled with an infrared excitation light source is an exciting modern imaging method that is enjoying widespread usage in the biomedical sciences. Thus, the present study was undertaken in order to evaluate the use of SHGM microscopy as a means to measure induced cross-linking effects in ex vivo rabbit sclera, following an injection of a chemical cross-linking agent into the sub-Tenon&#39;s space (sT), an injection approach that is standard practice for causing ocular anesthesia during ophthalmologic clinical procedures. The chemical cross-linking agent, sodium hydroxymethylglycinate (SMG), is from a class of cosmetic preservatives known as formaldehyde releasing agents (FARs). Scleral changes following reaction with SMG resulted in increases in SHG signals and correlated with shifts in thermal denaturation temperature, a standard method for evaluating induced tissue cross-linking effects.

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking TXL for Myopia

This protocol describes techniques for evaluating chemical cross-linking of the rabbit sclera using&#160;second harmonic generation imaging and differential scanning calorimetry.

Segunda geração harmônica sinais na esclera coelho como uma ferramenta para avaliação do tecido terapêutico do cross-linking (TXL) para miopia

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy ...