Somos gratos ao laboratório do Dr. Robert S. Kerbel (University of Toronto, Instituto de Pesquisa Sunnybrook, Toronto, Canadá) para obter ajuda técnica e experiência no desenvolvimento deste processo. Também gostaríamos de agradecer ao Dr. Sandra Sexton eo Roswell Park Departamento de Recursos Animais de Laboratório do Instituto do Câncer. Este trabalho foi apoiado por um prêmio da Fundação Aliança Roswell Park (para JMLE).

1. Rim ortotópico Implantação do Tumor <ol><li> Cultura de Células <ol><li> Antes da implantação ortotópico, cultivar células do rato RENCA LUC como uma monocamada a 75% de confluência. </li><li> Seguindo tripsinização e re-suspensão em 5% de FBS contendo meio, as células de centrifugação a 1.000 rpm, 4 ° C durante 5 minutos, repetindo três vezes para lavar em PBS. Em seguida, voltar a suspender as células em meio isento de soro para uma concentração de 5 x 10 4 RENCA LUC / 5 meios livres de soro ul. Nota: Dependendo da variante, ou linha celular utilizada, as células podem ser ressuspendidas numa proporção de matrigel e meio livre de soro de 1:1 a retardar o crescimento de células do rato altamente agressivo e difusão após a implantação. </li></ol></li><li> Cirurgia e implantação de células Nota: Todos os estudos com animais aqui descritos, incluindo a manutenção e determinação de terminais experimentais, foram realizados de acordo com um Comitê Animal Care e Use Institucional (IACUC) protocolo aprovado pelo Roswell Park Cancer Institute. <ol><li> Anestesiar um rato Balb / C utilizando isoflurano (2-3%). Aperte o pé e verificar se há reflexo para garantir anesthetization suficiente. Aplicar veterinário pomada para os olhos para evitar a secagem. Coloque o mouse em decúbito lateral direito e fazer a barba do lado esquerdo entre fore-e traseiras-membro. Aplicar álcool e iodo ao longo da área dorsal lombar para preparar assepticamente área de incisão. </li><li> Use uma tesoura cirúrgica para iniciar uma incisão de pele um centímetro no sentido longitudinal entre a última costela ea articulação do quadril. Solte o tecido conjuntivo sob a pele usando uma tesoura de dissecção romba. Use tesouras cirúrgicas para fazer incisão de 0,5 cm na direcção longitudinal na parede abdominal. Use uma pinça curva para empurrar suavemente para baixo em torno de ferida aberta - isso vai exteriorizar o rim e permitem a imobilização suave do órgão antes da injeção. </li><li> Coloque a seringa de Hamilton com 5 uL da mistura de células preparada. Para implante sub-capsularesções, dois métodos de implantação podem ser empregados: <ol><li> Superficial: Em paralelo com o rim, a agulha de inserção orientada longitudinalmente sob a cápsula renal (mas acima do parênquima) com a borda chanfrada para cima. Uma vez que a agulha está no lugar, injetar todas as células, até formas pequenas bolhas brancas. Lentamente, retire a agulha da cápsula e apagar imediatamente sobre injeção com um algodão aplicador derrubado estéril para absorver qualquer vazamento e evitar a disseminação de células. </li><li> Interno: Com borda chanfrada para cima e a partir do lado oposto do rim definitivo ao local de implantação, inserir a agulha através do interior do rim, até agulha é visível (mas não perfurar) o espaço sub-capsular. Injetar todas as células, até formas de bolhas brancas e local da injeção cotonete para evitar fugas. </li></ol></li><li> Suavemente retornar o rim para a cavidade do corpo. Feche a parede abdominal do corpo usando sutura absorvível 5-0 Vicryl. Uma vez completo, puxar a camada de pele em conjunto e colocar 2-3 wound clips. Assegurar clips são firmes e uniformemente espaçados para garantir que não haja exposição ferida. Antes de se administrar a recuperação de 500 ul de NaCl a 0,9% e 100 ul de buprenorfina (0,01 mg / mL) por via subcutânea utilizando uma agulha G 25. Rotineiramente monitorar colocação clipe e estabilidade, e retire depois de 10 dias. </li></ol></li></ol> 2. Nephrectomy / remoção do tumor primário <ol><li> Siga o protocolo idêntico para anesthetization mouse, imobilização, incisão e exposição do rim, conforme descrito nas seções 1.2.1 e 1.2.2 (acima). </li><li> Usar um segundo par de fórceps para isolar o rim suavemente a remoção do tecido de gordura de ligação da extremidade caudal e da glândula supra-renal a partir da extremidade craniana do rim. Ao segurar delicadamente o rim, use absorvível sutura Vicryl 5-0 para fazer um nó duplo ao redor do ureter, artéria renal e veia. Lentamente, seguro e, em seguida, cortada por cima do nó para remover o rim. Se qualquer sinal de perda de sangue imediatamente, ou se houver um risco de retenção insuficientede sutura no ureter, artéria e veia, então use cauterizador em vez de tesoura para cortar acima nó. </li><li> Uma vez rim é removido, verifique cuidadosamente para qualquer sangramento da artéria amarrado e usar cauterizador se necessário. Feche a parede abdominal do corpo usando Vicryl 5-0. Puxe a camada de pele em conjunto e colocar 2-3 clips ferida após mesmas precauções, conforme descrito no 1.2.4. Acompanhar de perto animais diariamente após cirurgia para sinais de sofrimento, sangramento, ou mobilidade limitada. Siga as orientações institucionais para animais com segurança. </li></ol> 3. Monitoramento metastático Progressão e localização em Endpoint <ol><li> Quantificação doença metastática: monitoramento Bioluminescent <ol><li> Ver metodologia previamente descrita na JOVE para monitorar metástase usando células transfectadas com luciferase e quantificados utilizando o sistema de imagem 11,12 Xenogen IVIS. </li></ol></li><li> Guia Visual necropsia para avaliação comparativa de distribuição metastáticoção <ol><li> Sacrificar animais de acordo com as diretrizes institucionais, utilizando endpoints, incluindo sinais de angústia, dificuldade para respirar, perda de peso, etc Nota: Fechar o monitoramento de animais é fundamental como progressão metastática espontânea pode ser altamente variável e rápida. </li><li> Use uma tesoura cirúrgica para iniciar uma incisão acima da uretra, que continua até a cavidade torácica a mandíbula inferior. Use uma tesoura sem corte-ended para separar a pele do músculo da parede abdominal. </li><li> Iniciado incisões ao longo dos braços e pernas. Peel pele de distância. Examine cuidadosamente a pele e fáscia da parede abdominal para nódulos, em seguida, os linfonodos superficiais: inguinal, braquial, axilar e cervical superficial. </li><li> Faça uma incisão na parede abdominal e corte ao longo das laterais esquerda e direita, até o diafragma. Com um corte horizontal remover a parte frontal da parede abdominal a nu as vísceras. </li><li> Examine a aparência imperturbável das vísceras fou grosseiramente tumores notáveis. Além disso, note o conteúdo do estômago e dos intestinos; se eles estão vazios, o rato pode não ter sido capaz de comer. Se o conteúdo for escuro, isto pode indicar evidência de hemorragia gastrointestinal superior / inferior. </li><li> Visualmente marcar cada órgão para a presença ou ausência de tumor, a atribuição de uma pontuação total para cada grupo de rato (4 animais por grupo é usado como exemplo na Figura 1D-i). Notas: 1) alargamento dos gânglios linfáticos, não significa necessariamente que é um crescimento do tumor; se é muito porte, muito firme, e uma cor esbranquiçada, é provável um nódulo metastático. 2) leitoso excessivo ou líquido turvo no abdômen indica ascite. Ascite podem incluir sólidos acumulações nos órgãos abdominais, particularmente entre os lóbulos do fígado e do estômago. 3) para os órgãos, tais como os pulmões, nódulos múltiplos podem estar presentes e contadas para comparações entre animais individuais (ver 13, por exemplo). </li><li> Identificar tumores brutas ou anormalidades dos órgãos abdominais individuais: fígado, baço, rins e pâncreas, removendo cuidadosamente e lavagem com PBS a partir de uma garrafa de esguicho. </li><li> Depois de retirar os órgãos abdominais, inspecionar os linfonodos abdominais, incluindo lombar, sacral, renal e linfonodos mesentéricos. </li><li> Avaliar diafragma para a evidência de metástase. Nota: Ampla propagação sobre o diafragma pode fazer com que perca sua contratilidade, levando ao trabalhado ou perda da respiração. </li><li> Remover diafragma e fazer duas incisões laterais na cavidade torácica, retirando a parte frontal para expor os pulmões eo coração. </li><li> Remover pulmões e coração delicadamente segurando e levantando o esôfago eo corte na traquéia acima do coração. Avaliar tecidos para os nódulos. Esprema coração. A falta de consistência elástica pode indicar presente tumor. </li><li> Use uma tesoura cirúrgica para cortar linearmente através do crânio. Remova cuidadosamente pequenos pedaços de crânio até ttodo ele cérebro está exposto. Olhe ao longo do cérebro para nódulos visíveis ou anomalias descolorações. </li></ol></li></ol>

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Os autores não têm nada a revelar.

O objectivo deste protocolo é o de avaliar a doença metastática espontâneo clinicamente relevantes usando um modelo do rato tumor singênico para descrever técnicas de implantação / ressecção. Actualmente, a maioria dos estudos pré-clínicos de novas terapêuticas experimentais avaliando não incluir o estudo de doença metastática e apenas poucos recapitulam as fases de crescimento do tumor primário, a ressecção cirúrgica, e eventual metástase espontânea. Até o momento, modelos de ratos geneticamente ...

A causa principal de mortalidade em pacientes com carcinoma de células renais (RCC) é a doença metastática sistémica que ocorre normalmente depois da remoção cirúrgica de um tumor primário em crescimento nos rins. No entanto, muito poucos modelos de tumores pré-clínicos que avaliaram terapêutica experimental em ratos incluem doença metastática, e menos ainda recapitular fielmente os estágios clínicos de crescimento localizado, cirurgia e iniciação micro-metástases espontânea e progressão 1-3. Esta diferença nos testes tornou-se cada vez mais importante na avaliação de novas terapias como às vezes impressionantes efeitos anti-tumorais observados em modelos animais nem sempre traduzidos para o tratamento da mesma forma bem-sucedida de pacientes 4. Tais diferenças nos resultados pode decorrer de eficácias de drogas diferenciais entre os modelos de tumor localizado ectópica ou ortotópico primárias e em estágio final de doença metastática 5-7. No caso da RCC, poucos estudos têm empregado estabeleceu umnimal protocolos que incluem a doença recorrente espontânea que imita os pacientes que têm, tipicamente, tinham rins portadores de tumor total ou parcialmente removidos de 2,3. As razões para essa falta de testes de modelo de rato variar. Em primeiro lugar, há o custo elevado dos animais e variabilidade inerente de selecção de células de tumor e o potencial metastático. Por exemplo, as linhas de células renais humanas tendem a raramente metástase, e devem ser selecionados ao longo de várias rodadas de implantação primário ortotópico e seleção metastático para derivar variantes que sempre divulgar e formam lesões distantes (ver descrição de tal linha celular uma derivação humana 8-10) . Por outro lado, células de ratos em modelos imunocompetentes tendem a se comportar de forma agressiva, e os números de celulares de baixa deve ser injetado com matrigel para reduzir a disseminação sistêmica imediato 3. Em segundo lugar, as dificuldades técnicas na realização de implantação adequada, a ressecção cirúrgica (nefrectomia) e rastreamento (e quantificação) metastati espontâneac crescimento pode ser desafiador e várias variáveis ​​críticas precisam de consideração quando se emprega essa técnica (veja a discussão para mais detalhes). O objectivo deste protocolo é o de descrever os passos essenciais (bem como armadilhas potenciais) de implantação ortotópico, a ressecção (nefrectomia), e monitoramento da doença RCC metastático espontânea e para oferecer uma orientação para padronizada (e mais generalizada) use entre laboratórios científicos que avaliar a eficácia de terapêuticas experimentais.

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		<col />
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		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">DMEM-high glucose with Pyr. And L-Glutamine</td>
			<td style="width:147px;">Corning</td>
			<td style="width:119px;">10-013-CV</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">FBS</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:119px;">10437-028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">0.25% Trypsin EDTA</td>
			<td style="width:147px;">Corning</td>
			<td style="width:119px;">25-053-CL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">1x DPBS without Calcium &#38; Magnesium</td>
			<td style="width:147px;">Corning</td>
			<td style="width:119px;">21-031-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Matrigel</td>
			<td style="width:147px;">BD Biosciences</td>
			<td style="width:119px;">354234</td>
			<td>must be kept on ice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Artifical tears-lubricant opthalmic ointment</td>
			<td style="width:147px;">Akorn Animal Health</td>
			<td style="width:119px;">17478-162-35</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Pocket pro pet trimmer</td>
			<td style="width:147px;">Braintree scientific</td>
			<td style="width:119px;">CLP9931B</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:397px;">Alcohol swab</td>
			<td style="width:147px;">VWR</td>
			<td style="width:119px;">326895</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Betadine solution swab</td>
			<td style="width:147px;">VWR</td>
			<td style="width:119px;">67618-152-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MICRO DISSECTING sissors straight,blunt - 25mm blades - 4.5&#34;&#160;</td>
			<td style="width:147px;">Southpointe surgical</td>
			<td style="width:119px;">RS-5982</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Iris Forceps, serrated, curved, 10cm long</td>
			<td style="width:147px;">Kent scientific</td>
			<td style="width:119px;">INS15915</td>
			<td>need two of these</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">10&#181;l Hamilton syringe</td>
			<td style="width:147px;">Hamilton</td>
			<td style="width:119px;">7635-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">30G, 45 degree, RN needle</td>
			<td style="width:147px;">Hamilton</td>
			<td style="width:119px;">7803-07</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:397px;">Sterile cotton tipped appicator</td>
			<td style="width:147px;">VWR</td>
			<td style="width:119px;">10805-144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">High temperature cautery kit</td>
			<td style="width:147px;">Kent scientific</td>
			<td style="width:119px;">INS500392</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">5-0 coated Vicryl, conventional cutting needle&#160;</td>
			<td style="width:147px;">Ethicon</td>
			<td style="width:119px;">J834</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Reflex clip applier for 7mm clips</td>
			<td style="width:147px;">Kent scientific</td>
			<td style="width:119px;">INS500343</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Reflex clips, 7mm, non-sterile</td>
			<td style="width:147px;">Kent scientific</td>
			<td style="width:119px;">INS500344</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Removing forceps, 12cm lone</td>
			<td style="width:147px;">Kent scientific</td>
			<td style="width:119px;">INS500347</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">0.9% Sodium Chloride</td>
			<td style="width:147px;">Baxter Healthcare</td>
			<td style="width:119px;">2B1322</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Buprenorphine 0.01mg/mL</td>
			<td style="width:147px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">25G 5/8&#34; needle</td>
			<td style="width:147px;">VWR</td>
			<td style="width:119px;">BD305122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">1mL syringe w/out needle</td>
			<td style="width:147px;">VWR</td>
			<td style="width:119px;">BD309659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">D-Luciferin</td>
			<td style="width:147px;">Gold Bio technology</td>
			<td style="width:119px;">LUCK-1G</td>
			<td></td>
		</tr>
	</tbody>
</table>

modeling spontaneous metastatic renal cell carcinoma (mrcc) in mice following nephrectomy

Um dos desafios chave para melhorar o teste de novas terapias experimentais em carcinoma de células renais (CCR) é o desenvolvimento de modelos que recapitulam fielmente evolução precoce e tardia da fase da doença metastática. Modelos de implantação do tumor típicos utilizar implantação do tumor primário ectópica ou ortotópico, mas poucos incluem doença metastática espontânea sistêmica que imita o ambiente clínico. Este protocolo descreve os principais passos para desenvolver RCC progressão da doença estágios semelhantes aos pacientes. Primeiro, ela usa um mouse linha de células tumorais altamente metastático em um modelo singênico para mostrar a implantação de células tumorais ortotópico. Os métodos incluem a implantação superficial e interna para o espaço sub-capsular com células combinadas com matrigel a evitar fugas e disseminação precoce. Em seguida, ele descreve os procedimentos para a excisão do rolamento de tumor de rim (nefrectomia), com crítica cuidados pré e pós-cirúrgico mouse. Finalmente, descreve as etapas necessárias para monitorar e avaliarmicro e macro-evolução da doença metastática, incluindo a imagem de bioluminescência também fornece um guia de necropsia visual detalhada para marcar distribuição da doença sistêmica. O objetivo desta descrição protocolo é facilitar o uso generalizado de modelos RCC metastático clinicamente relevantes para melhorar o valor preditivo do futuro teste terapêutico.

Figura 1A mostra um esquema que define os procedimentos detalhados neste resumo protocolo. Vários fatores importantes devem ser considerados para cada etapa. Por exemplo, no Passo 1 que mostra dois métodos para a implantação de células de tumor sub-capsular para o rim. As células tumorais podem ser implantados no espaço sub-capsular com um pequeno branco-bolha confirmando a colocação localizada de células com fugas impedido por remoção cuidadosa da agulha e limpando o excesso de escape de fl...

Watch this Scientific Journal Video about Modeling Spontaneous Metastatic Renal Cell Carcinoma mRCC in Mice Following Nephrectomy at JoVE.com

Modeling Spontaneous Metastatic Renal Cell Carcinoma mRCC in Mice Following Nephrectomy

Todos os modelos de metástase espontânea do carcinoma de células renais (RCC), a progressão da doença podem ser utilizados para avaliar os tratamentos em um ambiente clinicamente relevante. Este protocolo demonstra procedimentos diferentes para a implantação de células tumorais renal ortotópico, nefrectomia adequada, e, finalmente, apresenta um guia de necropsia para avaliação visual e bioluminescente de carga metastático e localização.

Modelagem espontânea metastático carcinoma de células renais (mRCC) em ratos após nefrectomia

One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully ...

modeling-spontaneous-metastatic-renal-cell-carcinoma-mrcc-mice

Research

JoVE Journal

Medicine

Modeling Spontaneous Metastatic Renal Cell Carcinoma (mRCC) in Mice Following Nephrectomy

16.5K visualizações.  Roswell Park Cancer Institute.  Todos os modelos de metástase espontânea do carcinoma de células renais (RCC), a progressão da doença podem ser utilizados para avaliar os tratamentos em um ambiente clinicamente relevante. Este protocolo demonstra procedimentos diferentes para a implantação de células tumorais renal ortotópico, nefrectomia adequada, e, finalmente, apresenta um guia de necropsia para avaliação visual e bioluminescente de carga metastático e localização. 

Vídeo: Modeling Spontaneous Metastatic Renal Cell Carcinoma mRCC in Mice Following Nephrectomy

Single Port Donor Nephrectomy

In 2007, Rane presented the first single port nephrectomy for a small non-functioning kidney at the World Congress of Endourology. Since that time, the use of single port surgery for nephrectomy has expanded to include donor nephrectomy. Over the next two years the technique was adopted for many others types of nephrectomies to include donor nephrectomy. We present our technique for single port donor nephrectomy using the Gelpoint device. We have successfully performed this surgery in over 100 patients and add this experience to our experience of over 1000 laparoscopic nephrectomies. With the proper equipment and technique, single port donor nephrectomy can be performed safely and effectively in the majority of live donors. We have found that our operative times and most importantly our transplant outcomes have not changed significantly with the adoption of the single port donor nephrectomy. We believe that single port donor nephrectomy represents a step forward in the care of living donors.

Single port laparoscopic surgery is changing the standard of care in surgical care like nothing since the laparoscopic technique was introduced 20 years ago. We present out technique of single port donor nephrectomy using the Gelpoint device. We have successfully performed this surgery in 100 patients.

Nefrectomia Single Port

In 2007, Rane presented the first single port nephrectomy for a small non-functioning kidney at the World Congress of Endourology.  Since that time, the ...

Medicina

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

Curso de modelagem em Ratos - Oclusão da Artéria Cerebral Média com o modelo de Filamento

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

Mammary Transplantation of Stromal Cells and Carcinoma Cells in C57BL/6J Mice

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular through transplantation of stromal cells and epithelial cells in the mammary gland of mice. Current transplantation models often involve the use of immunocompromised mice due to the different genetic backgrounds of stromal cells and epithelial cells. Extracellular matrices are often used to embed the two different cell types for consistent cell-cell interactions, but involve the use of Matrigel or rat tail collagen, which are immunogenic substrates. The lack of functional T cells from immunocompromised mice prevents accurate assessment of stromal cells on mammary tumor progression in vivo, with important implications on drug development and efficacy. Moreover, immunocompromised mice are costly, hard to breed and require special care conditions. To overcome these obstacles, we have developed an approach to orthotopically transplant stromal cell and epithelial cells into mice from the same genetic background to induce consistent tumor formation. This system involves harvesting normal, carcinoma associated fibroblasts, PyVmT mammary carcinoma cells and collagen from donor C57BL/6J mice. The cells are then embedded in collagen and transplanted in the inguinal mammary glands of female C57BL/6J mice. Transplantation of PyVmT cells alone form palpable tumors 30-40 days post transplantation. Endpoint analysis at 60 days indicates that co-transplantation with fibroblasts enhances mammary tumor growth compared to PyVmT cells transplanted alone. While cells and matrix from C57BL/6J mice were used in these studies, the isolation of cells and matrix and transplantation approach may be applied towards mice from different genetic backgrounds demonstrating versatility. In summary, this system may be used to investigate molecular interactions between stromal cells and epithelial cells, and overcomes critical limitations in immunocompromised mouse models.

In this report, we demonstrate a system to isolate and culture donor cells from the mouse mammary gland, and orthotopically transplant these cells in recipient mice to analyze stromal: epithelial interactions during mammary tumor development.

Transplante mamária de células estromais e células de carcinoma em camundongos C57BL/6J

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular ...

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 ...

Modeling Intracerebral Hemorrhage in Mice: Injection of Autologous Blood or Bacterial Collagenase

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related hospitalizations and for which no effective treatments are available to date1,2. Because of the heterogeneity of ICH in humans, various preclinical models are needed to thoroughly explore prospective therapeutic strategies3. Experimental ICH is commonly induced in rodents by intraparenchymal injection of either autologous blood or bacterial collagenase4. The appropriate model is selected based on the pathophysiology of hemorrhage induction and injury progression. The blood injection model mimics a rapidly progressing hemorrhage. Alternatively, bacterial collagenase enzymatically disrupts the basal lamina of brain capillaries, causing an active bleed that generally evolves over several hours5. Resultant perihematomal edema and neurofunctional deficits can be quantified from both models. In this study, we described and evaluated a modified double injection model of autologous whole blood6 as well as an ICH injection model of bacterial collagenase7, both of which target the basal ganglia (corpus striatum) of male CD-1 mice. We assessed neurofunctional deficits and brain edema at 24 and 72 hr after ICH induction. Intrastriatal injection of autologous blood (30 &mu;l) or bacterial collagenase (0.075U) caused reproducible neurofunctional deficits in mice and significantly increased brain edema at 24 and 72 hr after surgery (p&lt;0.05). In conclusion, both models yield consistent hemorrhagic infarcts and represent basic methods for preclinical ICH research.

Clinically relevant animal models of intracerebral hemorrhage (ICH) are needed to extend our knowledge of hemorrhagic stroke and to examine novel therapeutic strategies. In this study, we describe and evaluate two ICH models that implement unilateral injections of either autologous whole blood or bacterial collagenase into the basal ganglia (corpus striatum) of mice.

Hemorragia modelagem intracerebral em camundongos: Injeção de sangue autólogo ou colagenase bacteriana

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related ...

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  ...

Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice:  A Model for Immunotherapy Development

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy and/or cytotoxic chemotherapy. To induce bladder cancer, C57BL/6 mice (MUC1.Tg and wild type) were treated orally with the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN) at 3.0 mg/day, 5 days/week for 12 weeks. To assess the effects of OH-BBN on serum cytokine profile during tumor development, whole blood was collected via submandibular bleeds prior to treatment and every four weeks. In addition, a MUC1-targeted peptide vaccine and placebo were administered to groups of mice weekly for eight weeks. Multiplex fluorometric microbead immunoanalyses of serum cytokines during tumor development and following vaccination were performed. At termination, interferon gamma (IFN-&#947;)/interleukin-4 (IL-4) ELISpot analysis for MUC1 specific T-cell immune response and histopathological evaluations of tumor type and grade were performed. The results showed that: (1) the incidence of bladder cancer in both MUC1.Tg and wild type mice was 67%; (2) transitional cell carcinomas (TCC) developed at a 2:1 ratio compared to squamous cell carcinomas (SCC); (3) inflammatory cytokines increased with time during tumor development; and (4) administration of the peptide vaccine induces a Th1-polarized serum cytokine profile and a MUC1 specific T-cell response. All tumors in MUC1.Tg mice were positive for MUC1 expression, and half of all tumors in MUC1.Tg and wild type mice were invasive. In conclusion, using a team approach through the coordination of the efforts of pharmacologists, immunologists, pathologists and molecular biologists, we have developed an immune intact transgenic mouse model of bladder cancer that expresses hMUC1.

An N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder cancer model was developed in human mucin 1 (MUC1) transgenic mice for the purpose of testing MUC1-directed immunotherapy. After administering a MUC1-targeted peptide vaccine, a cytotoxic T lymphocyte response to MUC1 was confirmed by measuring serum cytokine levels and T-cell specific activity.

Indução de carcinoma da bexiga invasivo de células transicionais em imunológico intacto Humanos MUC1 Ratos transgênicos: um modelo de desenvolvimento Imunoterapia

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

Início do Carcinoma de mama metastático por Segmentação do ductal Epithelium com Adenovírus-Cre: Um Modelo Novel de camundongo transgênico do Câncer de Mama

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical options are available for a small proportion of stroke patients in the acute phase. Current research is intensively searching for novel therapeutic strategies and is increasingly focusing on the sub-acute and chronic phase after stroke because more patients might be eligible for therapeutic interventions in a prolonged time window. These delayed mechanisms include important pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity and regeneration. In order to analyze these mechanisms and to subsequently evaluate novel drug targets, experimental stroke models with clinical relevance, low mortality and high reproducibility are sought after. Moreover, mice are the smallest mammals in which a focal stroke lesion can be induced and for which a broad spectrum of transgenic models are available. Therefore, we describe here the mouse model of transcranial, permanent coagulation of the middle cerebral artery via electrocoagulation distal of the lenticulostriatal arteries, the so-called &#8220;coagulation model&#8221;. The resulting infarct in this model is located mainly in the cortex; the relative infarct volume in relation to brain size corresponds to the majority of human strokes. Moreover, the model fulfills the above-mentioned criteria of reproducibility and low mortality. In this video we demonstrate the surgical methods of stroke induction in the &#8220;coagulation model&#8221; and report histological and functional analysis tools.

Various murine models of middle cerebral artery occlusion (MCAO) are widely used in experimental brain research. Here, we demonstrate the model of transcranial permanent distal MCAO which produces consistent cortical infarction of a size corresponding to damage imposed by the majority of human ischemic strokes.

Curso de modelagem em Ratos: Coagulação permanente da artéria distal Cerebral Média

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical ...

Modeling Chemotherapy Resistant Leukemia In Vitro

It is well established that the bone marrow microenvironment provides a unique site of sanctuary for hematopoietic diseases that both initiate and progress in this site. The model presented in the current report utilizes human primary bone marrow stromal cells and osteoblasts as two representative cell types from the marrow niche that influence tumor cell phenotype. The in vitro co-culture conditions described for human leukemic cells with these primary niche components support the generation of a chemoresistant subpopulation of tumor cells that can be efficiently recovered from culture for analysis by diverse techniques. A strict feeding schedule to prevent nutrient fluxes followed by gel type 10 cross-linked dextran (G10) particles recovery of the population of tumor cells that have migrated beneath the adherent bone marrow stromal cells (BMSC) or osteoblasts (OB) generating a "phase dim" (PD) population of tumor cells, provides a consistent source of purified therapy resistant leukemic cells. This clinically relevant population of tumor cells can be evaluated by standard methods to investigate apoptotic, metabolic, and cell cycle regulatory pathways as well as providing a more rigorous target in which to test novel therapeutic strategies prior to pre-clinical investigations targeted at minimal residual disease.

Modeling Chemotherapy Resistant Leukemia In Vitro

The current report summarizes a protocol that can be utilized to model the influence of the bone marrow microenvironment niche on leukemic cells with emphasis placed on enrichment of the most chemoresistant subpopulation.