KF e KO são co primeiros autores, juntamente com KS Este trabalho foi financiado em parte pelo AHPBA Fellowship (KS), NIH K23 CA148964-01 (LZ), Johns Hopkins School of Medicine Clinical Scientist Award (LZ), Fundação Viragh e do Centro de Câncer Ir Viragh pâncreas na Universidade Johns Hopkins (EMJ e LZ), a Fundação Nacional de Pâncreas (LZ), o Lefkofsky Família Foundation (LZ), o SPORE NCI em Gastrointestinal Cânceres P50 CA062924 (EMJ, LZ), a Fundação Lustgarten (LZ), e as bolsas Sol Goldman pâncreas Cancer Center (KS , BHE, LZ).

Todos os experimentos com animais conformados com as orientações do Comitê de Cuidados e Uso da Universidade Johns Hopkins animal, e os animais foram mantidos de acordo com as diretrizes da Associação para a Avaliação e Acreditação do Laboratório de Cuidados (AAALAC). O procedimento cirúrgico é realizado sob condições estéreis, numa sala de operações, que passa por um mínimo de quinze trocas de ar por hora. Todos os instrumentos são esterilizados em autoclave antes do procedimento e novamente com etanol a 70%, entre cada um dos ratinhos durante o procedimento. Todos os ratos que não se espera para sobreviver ao procedimento cirúrgico são sacrificados sob CO 2 durante 3 min, seguido por deslocação cervical, enquanto ainda sob anestesia. 1. celular Preparação <ol><li> Ressuspender as células em cultura in Panc02 salina tamponada com fosfato a 2 x10 7 células / ml, e manter em gelo. </li></ol> 2. Rato Preparação <ol><li> Administrar cetamina-E (100 mg / kg) misturado com xilazina (10 mg / kg) por via intraperitoneal com 8-12 semanas de idade C57BL / 6 fêmeas, em doses divididas. </li><li> Verifique se o dedo do pé retirada pitada reflexo é abolido para garantir que o mouse é totalmente anestesiado. </li><li> Administrar doses suplementares de cetamina (100 mg / kg) misturado com xilazina (10 mg / kg), conforme necessário para anestesiar completamente os ratinhos. </li><li> Aplicar Puralube Vet Pomada para os olhos dos camundongos para evitar a secagem quando os ratos estão sob anestesia. </li><li> Raspar a área cirúrgica dos ratinhos a fim de evitar a contaminação da ferida. </li><li> Prep a área subcostal esquerda da incisão com etanol a 70% e depois de iodo. </li><li> Coloque um campo cirúrgico em torno do abdômen para manter a esterilidade. </li></ol> 3. Laparotomia <ol><li> Fazer uma incisão subcostal esquerda em linha com a orelha esquerda através da pele e através do peritoneu utilizando um bisturi. </li><li> Expresse o baço através da incisão através da aplicação p digitais simultânearessão ao longo dos aspectos cranial e caudal da incisão. </li><li> Localizar os vasos sanguíneos do baço na extremidade inferior do baço. </li><li> Divida o baço, colocando dois Horizonte médias clips ligadura no centro do baço tomando cuidado para não danificar os vasos sanguíneos do baço nos pólos do baço. </li><li> Inserir o pólo superior do baço de volta para o peritoneu para evitar a contaminação. </li></ol> 4 Tumor Injeção <ol><li> Desenha-se 150 ul de solução salina tamponada com fosfato para um 26 L x 5/8 &quot;seringa. </li><li> Elaborar 100 l de Panc02 (2 x10 6) células para a mesma seringa manter a seringa na posição vertical em todos os momentos durante a injecção. </li><li> Mergulhar a agulha numa solução de etanol a 70%. A agulha é imersa em etanol a 70% para assegurar a esterilidade. </li><li> Injetar as células lentamente no hemispleen exposto tendo a certeza da seringa é mantido na vertical em todos os momentos. O bisel da syringe devem ser observados em todos os momentos através da cápsula do baço para evitar a injeção das células sob o baço. A clareamento dos vasos baço e de sangue devem ser observados após a injecção. </li><li> Eleve o baço e aplicar um meio Horizon clipe sob o baço para ocluir os vasos sanguíneos do baço. </li><li> Aplicar um pequeno clipe Horizon para o aspecto mais distal do pâncreas e vasos esplênicos. </li><li> Ligadura do pâncreas e vasos esplênicos da hemispleen cortando acima dos clips horizonte. </li><li> Posicione o mouse sobre o seu &#39;lado, e irrigar a incisão com água destilada. </li></ol> 5. Abdominal Encerramento e recuperação da anestesia <ol><li> Feche o peritônio com um ponto 4-0 em execução. </li><li> Aplicar 2-3 clips de pele para fechar a incisão na pele. </li><li> Administrar 0,1 mg / kg de buprenorfina ou 5-10 mg / kg por via subcutânea Carprofen para aliviar a dor pós-cirúrgica. </li><li> Posicione o mouse sobre uma almofada de aquecimento para recovery até celular eo mouse demonstra padrões respiratórios regulares. Os animais só são devolvidos para a companhia de outros animais depois de se recuperar totalmente do procedimento cirúrgico. </li></ol> 6. Necropsia Exame e colheita do Fígado <ol><li> Entre 30 a 60 dias após a cirurgia, os ratos começam a mostrar sintomas clínicos de metástase e vai exigir a eutanásia. Sinais de doença metastática que requerem a eutanásia humanitária incluem abdomens dilatados eo desenvolvimento de ascite. Eutanásia ratos por inalação de CO2. </li><li> Após a inalação, realizar deslocamento cervical. </li><li> Depois que o animal tenha sido determinada a ser não responsivo, fazer uma incisão através do peritônio para expor o abdômen com uma tesoura. </li><li> Utilizando uma pinça e uma tesoura, cortou delicadamente o fígado para fora do abdômen. </li><li> Coloque o fígado em composto outubro, eo flash congelar usando nitrogênio líquido. Armazenar o fígado a -80 ° C. Alternativamente, o ao vivor pode ser fixado em 16% de formalina neutra tamponada a TA durante 24 h e embebido em parafina. </li></ol>

<ol>
	<li>American Cancer Society. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cancer Facts &amp; Figures. , (2013).</li><li>Clark, C. E., Beatty, G. L., Vonderheide, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunosurveillance+of+pancreatic+adenocarcinoma:+insights+from+genetically+engineered+mouse+models+of+cancer.">Immunosurveillance of pancreatic adenocarcinoma: insights from genetically engineered mouse models of cancer.</a> Cancer letters. 279, 1-7 (2009).</li><li>Rhim, A. 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R., 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=Trp53R172H+and+KrasG12D+cooperate+to+promote+chromosomal+instability+and+widely+metastatic+pancreatic+ductal+adenocarcinoma+in+mice.">Trp53R172H and KrasG12D cooperate to promote chromosomal instability and widely metastatic pancreatic ductal adenocarcinoma in mice.</a> Cancer cell. 7, 469-483 (2005).</li><li>Marion, A., Aoudi, W., Basarab, A., Delachartre, P., Vray, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+flow+evaluation+in+high-frequency,+40+MHz+imaging:+a+comparative+study+of+four+vector+velocity+estimation+methods.">Blood flow evaluation in high-frequency, 40 MHz imaging: a comparative study of four vector velocity estimation methods.</a> Ultrasonics. 50, 683-690 (2010).</li><li>Zheng, L., 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=Tyrosine+23+phosphorylation-dependent+cell-surface+localization+of+annexin+A2+is+required+for+invasion+and+metastases+of+pancreatic+cancer.">Tyrosine 23 phosphorylation-dependent cell-surface localization of annexin A2 is required for invasion and metastases of pancreatic cancer.</a> PloS one. 6, e19390 (2011).</li></ol>

Os autores não têm conflitos de interesse relevantes para divulgar.

Este modelo murino pré-clínico de metástase do câncer pancreático para o fígado através de uma técnica de injeção hemispleen é o primeiro modelo descrito que espelha muitas das condições clínicas e imunológicas de pacientes com doença metastática para o fígado. Este modelo apresenta várias vantagens em relação aos outros modelos de murino. Em primeiro lugar, ao contrário dos modelos transgénicos de cancro pancreático, em que apenas 30% dos ratinhos desenvolvem metástases do fígado e a temporiza...

Adenocarcinoma ductal pancreático (PDA) é a quarta principal causa de mortes relacionadas ao câncer nos Estados Unidos 1. A sobrevida em cinco anos para pacientes com PDA metastático é 2-12% 1. Embora a quimioterapia adjuvante e neoadjuvante para câncer de pâncreas é mais eficaz do que nunca hoje em dia, ainda há uma necessidade desesperada de novas terapias. O desenvolvimento de novas terapêuticas necessita de modelos animais pré-clínicos fidedignos. Embora os modelos de tumores subcutâneos são simples de utilizar, desvantagens incluem a incapacidade destes tumores e a metástase imitar a progressão do tumor e microambientes visto em cancros humanos. Modelos geneticamente modificados, tais como o rato geneticamente modificadas contendo Kras e p53 knock-in mutações oncogénicas (o modelo KPC) 2 e o modelo KPC com um marcador de linhagem YFP (o modelo PKCY 3) permitem o estudo de tumores que ocorrem naturalmente em immunocompetecamundongos NT. Além disso, o micro-ambiente do tumor é inalterada e é mais parecida com a observada na progressão de tumores humanos. Infelizmente, o tempo de desenvolvimento do tumor é variável dentro desses modelos, o que torna difícil de estudar terapias experimentais. Além disso, backcrossing desses ratos requer uma quantidade significativa de tempo e recursos financeiros, o que nem sempre é viável. Por fim, os modelos de mouse xenotransplante PDA implantados tornem indispensável ratos, que foram alterados ou microambientes tumorais ausentes imunocomprometidos. Como resultado, a eficácia de terapias experimentais demonstraram nestes modelos pré-clínicos são muitas vezes não reprodutíveis em ensaios clínicos humanos 4. Este modelo de rato hemiesplenectomia utiliza células tumorais Panc02, que são um, metilcolantreno induzida linha celular de tumor pancreático altamente tumorigénicas derivadas de C57BL / 6 5, 6. Além disso, outras linhas celulares PDA murino derivado do mic KPCe pode ser utilizado neste modelo. Schulick et al. Já desenvolveu uma técnica hemiesplenectomia e criou um modelo singênico rato de metástases de câncer de cólon 7. Este modelo hemiesplenectomia (Figura 1) oferece a inoculação do tumor consistente e iguais em camundongos imunocompetentes prestando-se à investigação de novos agentes terapêuticos 7-10.

<table border="0" cellpadding="0" cellspacing="0" width="713">
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			<td height="42" style="height:42px;width:216px;">Cell culture media and components</td>
			<td style="width:197px;"></td>
			<td style="width:119px;"></td>
			<td style="width:181px;">will vary depending on cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Phosphate Buffered Saline</td>
			<td style="width:197px;">Gibco by Life Technologies</td>
			<td style="width:119px;">10010-023</td>
			<td style="width:181px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">15 mL centrifigure tubes</td>
			<td style="width:197px;">Corning</td>
			<td style="width:119px;">430052</td>
			<td style="width:181px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Curved Operating Scissor</td>
			<td style="width:197px;">Roboz Surgical Store</td>
			<td style="width:119px;">RS-6835</td>
			<td style="width:181px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Micro dissecting Graefe Forceps</td>
			<td style="width:197px;">Roboz Surgical Store</td>
			<td style="width:119px;">RS-5130</td>
			<td style="width:181px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Needle holder (regular)</td>
			<td style="width:197px;">World Precision Instruments, Inc</td>
			<td style="width:119px;"></td>
			<td style="width:181px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">3-0 or 4-0 suture</td>
			<td style="width:197px;"></td>
			<td style="width:119px;"></td>
			<td style="width:181px;">courtesy of dept of surgery, Johns Hopkins</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Weck Horizon Open Ligating Clip Applier, small, angled</td>
			<td style="width:197px;">Teleflex</td>
			<td style="width:119px;">137085</td>
			<td style="width:181px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Weck Horizon Open Ligating Clip Applier, medium, angled</td>
			<td style="width:197px;">Teleflex</td>
			<td style="width:119px;">237115</td>
			<td style="width:181px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Weck Horizon medium ligating clips</td>
			<td style="width:197px;">Teleflex</td>
			<td style="width:119px;">002200</td>
			<td style="width:181px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Weck Horizon small ligating clips</td>
			<td style="width:197px;">Teleflex</td>
			<td style="width:119px;">001200</td>
			<td style="width:181px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Syringe, 1 cc, tb syringe, 3/8&#34; slip tip</td>
			<td style="width:197px;">Becton Dickinson</td>
			<td style="width:119px;">309625</td>
			<td style="width:181px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Syringe, 1 cc, tb syringe, 5/8&#34; slip tip</td>
			<td style="width:197px;">Becton Dickinson</td>
			<td style="width:119px;">309597</td>
			<td style="width:181px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">9 mm Autoclip Applier</td>
			<td style="width:197px;">Mikron</td>
			<td style="width:119px;">427630</td>
			<td style="width:181px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">9 mm Autoclip</td>
			<td style="width:197px;">Becton Dickinson</td>
			<td style="width:119px;">427631</td>
			<td style="width:181px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Heating pad</td>
			<td style="width:197px;">Sunbeam</td>
			<td style="width:119px;">CAT93</td>
			<td style="width:181px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">70% Ethanol</td>
			<td style="width:197px;"></td>
			<td style="width:119px;"></td>
			<td style="width:181px;">sold by numerous vendors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ketamine Hydrochloride&#160;</td>
			<td style="width:197px;">Hospira</td>
			<td style="width:119px;">22395DD</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Xylazine hydrochloride</td>
			<td style="width:197px;">Sigma</td>
			<td style="width:119px;">X1251</td>
			<td></td>
		</tr>
	</tbody>
</table>

a preclinical murine model of hepatic metastases

Numerosos modelos murinos foram desenvolvidas para estudar os cancros humanos e avançar o entendimento de tratamento e o desenvolvimento do cancro. Aqui, um modelo pré-clínico, murino tumor pancreático de metástases hepáticas através de uma injecção de células tumorais hemispleen pancreáticos de murino singeneicas é descrito. Este modelo imita muitas das condições clínicas em pacientes com doença metastática para o fígado. Ratos consistentemente desenvolvem metástases no fígado, permitindo a investigação do processo metastático, testes terapia experimental, e a pesquisa da imunologia tumoral.

Células tumorais Panc02 injetados na hemispleen (Figura 1) formar metástases hepáticas em 100% dos ratos, enquanto metástases pulmonares e peritoneal não são observados se a técnica é realizada corretamente. Esta técnica foi realizada em mais de cem ratos usando células Panc02 em vários experimentos independentes, e reprodutível, todos os ratos não tratados morrem com metástases hepáticas entre 30 e 60 dias após o procedimento hemiesplenectomia (Figura 2). Para obter sig...

Watch this Scientific Journal Video about A Preclinical Murine Model of Hepatic Metastases at JoVE.com

A Preclinical Murine Model of Hepatic Metastases

A modelo pré-clínico, murino de metástases hepáticas realizado através de uma técnica de injeção hemispleen.

Um Modelo murino de metástases hepáticas pré-clínica

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, ...

a-preclinical-murine-model-of-hepatic-metastases

Research

JoVE Journal

Medicine

28.3K visualizações.  The Johns Hopkins University School of Medicine.  A modelo pré-clínico, murino de metástases hepáticas realizado através de uma técnica de injeção hemispleen. 

Vídeo: A Preclinical Murine Model of Hepatic Metastases

Murine Bioluminescent Hepatic Tumour Model

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are administered specifically to the liver to induce a localised liver tumour, via mobilisation of the spleen and splitting into two, leaving intact the vascular pedicle for each half of the spleen. Lewis lung carcinoma cells that constitutively express the firefly luciferase gene (luc1) are inoculated into one hemi-spleen which is then resected 10 minutes later. The other hemi-spleen is left intact and returned to the abdomen. Liver tumour growth can be monitored by bioluminescence imaging using the IVIS whole body imaging system. Quantitative imaging of tumour growth using IVIS provides precise quantitation of viable tumour cells. Tumour cell death and necrosis due to drug treatment is indicated early by a reduction in the bioluminescent signal. This mouse model allows for investigating the mechanisms underlying metastatic tumour-cell survival and growth and can be used for the evaluation of therapeutics of liver metastasis. 

This article describes a procedure for the induction of orthotopic bioluminescent liver tumours in mice, and subsequent analysis of tumour growth confined to the liver using live whole body luminescence imaging.

Modelo de tumor hepático murino Bioluminescent

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are ...

Medicina

Gene Transfer for Ischemic Heart Failure in a Preclinical Model

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine their efficacy and safety.
Gene therapy using viral vectors is one of the most promising approaches for treating cardiac diseases. Viral delivery of various different genes by changing the carrier gene has immeasurable therapeutic potential.
In this video, the full process of an animal model of heart failure creation followed by gene transfer is presented using a swine model. First, myocardial infarction is created by occluding the proximal left anterior descending coronary artery. Heart remodeling results in chronic heart failure. Unique to our model is a fairly large scar which truly reflects patients with severe heart failure who require aggressive therapy for positive outcomes. After myocardial infarct creation and development of scar tissue, an intracoronary injection of virus is demonstrated with simultaneous nitroglycerine infusion. Our injection method provides simple and efficient gene transfer with enhanced gene expression. This combination of a myocardial infarct swine model with intracoronary virus delivery has proven to be a consistent and reproducible methodology, which helps not only to test the effect of individual gene, but also compare the efficacy of many genes as therapeutic candidates.

A method of gene transfer for the treatment of ischemic heart failure is described using a swine model of myocardial infarction. Our simple and reproducible method enables us to readily evaluate the efficacy of various gene transfers with a very simple and reproducible way.

Transferência de genes para insuficiência cardíaca isquêmica em um modelo pré-clínica

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine ...

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

Modelo Murino de Cicatrização

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.

A standardized mouse model of subarachnoid hemorrhage by intraluminal Circle of Willis perforation is described. Vessel perforation and subarachnoid bleeding are monitored by intracranial pressure monitoring. In addition various vital parameters are recorded and controlled to maintain physiologic conditions.

Um modelo murino de hemorragia subaracnóide

In this video publication a standardized mouse model of subarachnoid  hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of  Willis ...

Murine Model for Non-invasive Imaging to Detect and Monitor Ovarian Cancer Recurrence

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of care consisting of surgical debulking and combination chemotherapy consisting of platinum and taxane compounds, almost 90% of patients recur within a few years. In these patients the development of chemoresistant disease limits the efficacy of currently available chemotherapy agents and therefore contributes to the high mortality. To discover novel therapy options that can target recurrent disease, appropriate animal models that closely mimic the clinical profile of patients with recurrent ovarian cancer are required. The challenge in monitoring intra-peritoneal (i.p.) disease limits the use of i.p. models and thus most xenografts are established subcutaneously. We have developed a sensitive optical imaging platform that allows the detection and anatomical location of i.p. tumor mass. The platform includes the use of optical reporters that extend from the visible light range to near infrared, which in combination with 2-dimensional X-ray co-registration can provide anatomical location of molecular signals. Detection is significantly improved by the use of a rotation system that drives the animal to multiple angular positions for 360 degree imaging, allowing the identification of tumors that are not visible in single orientation. This platform provides a unique model to non-invasively monitor tumor growth and evaluate the efficacy of new therapies for the prevention or treatment of recurrent ovarian cancer.

We describe a non-invasive animal imaging platform that allows the detection, quantification, and monitoring of ovarian cancer growth and recurrence. This intra-peritoneal xenograft model mimics the clinical profile of patients with ovarian cancer.

Modelo murino de imagem não invasivo para detectar e monitorar o cancro do ovário Recorrência

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of ...

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both direct and rapid analysis of therapeutic sensitivity and resistance. However, recent evidence suggests that therapeutic response is not exclusive to the inherent molecular composition of cancer cells but rather is greatly influenced by the tumor cell microenvironment, a feature that cannot be recapitulated by traditional culturing methods. Even implementation of tumor xenografts, though providing a wealth of information on drug delivery/efficacy, cannot capture the tumor cell/microenvironment crosstalk (i.e., soluble factors) that occurs within human tumors and greatly impacts tumor response. To this extent, we have developed an ex vivo (fresh tissue sectioning) technique which allows for the direct assessment of treatment response for preclinical and clinical therapeutics development. This technique maintains tissue integrity and cellular architecture within the tumor cell/microenvironment context throughout treatment response providing a more precise means to assess drug efficacy.

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

Established cancer cell lines and xenografts have been the mainstay of cancer research for the past several decades. However, recent evidence suggests that therapeutic response is greatly influenced by the tumor cell microenvironment. Therefore, we have developed an ex vivo analysis of primary tumor specimens for drug development purposes.

Ex Vivo resposta ao tratamento de tumores primários e / ou Associated Metástases para pré-clínica e clínica de Desenvolvimento Therapeutics

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both ...

Isolated Hepatic Perfusion as a Treatment for Liver Metastases of Uveal Melanoma

Isolated hepatic perfusion (IHP) is a procedure where the liver is surgically isolated and perfused with a high concentration of the chemotherapeutic agent melphalan. Briefly, the procedure starts with the setup of a percutaneous veno-venous bypass from the femoral vein to the external jugular vein. Via a laparotomy, catheters are then inserted into the proper hepatic artery and the caval vein. The portal vein and the caval vein, both supra- and infrahepatically, are then clamped. The arterial and venous catheters are connected to a heart lung machine and the liver is perfused with melphalan (1 mg/kg body weight) for 60 min. This way it is possible to locally perfuse the liver with a high dose of a chemotherapeutic agent, without leakage to the systemic circulation.
In previous studies including patients with isolated liver metastases of uveal melanoma, an overall response rate of 33-100% and a median survival between 9 and 13 months, have been reported. The aim of this protocol is to give a clear description of how to perform the procedure and to discuss IHP as a treatment option for liver metastases of uveal melanoma.

Here, we present a protocol how to perform an isolated liver perfusion (IHP) with melphalan and we also discuss IHP as a treatment option for liver metastases of uveal melanoma.

Isolado hepática Perfusão como um tratamento para o fígado Metástases de Uveal Melanoma

Isolated hepatic perfusion (IHP) is a procedure where the liver is surgically isolated and perfused with a high concentration of the chemotherapeutic ...

Percutaneous Hepatic Perfusion (PHP) with Melphalan as a Treatment for Unresectable Metastases Confined to the Liver

Unresectable liver metastases of colorectal cancer can be treated with systemic chemotherapy, aiming to limit the disease, extend survival or turn unresectable metastases into resectable ones. Some patients however, suffer from side effects or progression under systemic treatment. For patients with metastasized uveal melanoma there are no standard systemic therapy options. For patients without extrahepatic disease, isolated liver perfusion (IHP) may enable local disease control with limited systemic side effects. Previously, this was performed during open surgery with satisfying results, but morbidity and mortality related to the open procedure, prohibited a widespread application. Therefore, percutaneous hepatic perfusion (PHP) with simultaneous chemofiltration was developed. Besides decreasing morbidity and mortality, this procedure can be repeated, hopefully leading to a higher response rate and improved survival (by local control of disease). During PHP, catheters are placed in the proper hepatic artery, to infuse the chemotherapeutic agent, and in the inferior caval vein to aspirate the chemosaturated blood returning through the hepatic veins. The caval vein catheter is a double balloon catheter that prohibits leakage into the systemic circulation. The blood returning from the hepatic veins is aspirated through the catheter fenestrations and then perfused through an extra-corporeal filtration system. After filtration, the blood is returned to the patient by a third catheter in the right internal jugular vein. During PHP a high dose of melphalan is infused into the liver, which is toxic and would lead to life threatening complications when administered systemically. Because of the significant hemodynamic instability resulting from the combination of caval vein occlusion and chemofiltration, hemodynamic monitoring and hemodynamic support is of paramount importance during this complex procedure.

Percutaneous Hepatic Perfusion PHP with Melphalan as a Treatment for Unresectable Metastases Confined to the Liver

In this manuscript, we describe percutaneous isolated hepatic perfusion with simultaneous chemofiltration as treatment for unresectable liver metastases. This procedure is performed under general anaesthesia in the angiosuite by an experienced team, consisting of an interventional radiologist, a clinical perfusionist and anaesthesiologist.

Percutânea hepática Perfusão (PHP) com melfalano como um tratamento para Unresectable Metástases confinado ao fígado

Unresectable liver metastases of colorectal cancer can be treated with systemic chemotherapy, aiming to limit the disease, extend survival or turn ...

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 ...

Preclinical Model of Hind Limb Ischemia in Diabetic Rabbits

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular disease is the development of ischemia. In severe cases, patients can develop critical limb ischemia in which they experience constant pain and an increased risk of limb amputation. Current therapies for peripheral ischemia include bypass surgery or percutaneous interventions such as angioplasty with stenting or atherectomy to restore blood flow. However, these treatments often fail to the continued progression of vascular disease or restenosis or are contraindicated due to the overall poor health of the patient. A promising potential approach to treat peripheral ischemia involves the induction of therapeutic neovascularization to allow the patient to develop collateral vasculature. This newly formed network alleviates peripheral ischemia by restoring perfusion to the affected area. The most frequently employed preclinical model for peripheral ischemia utilizes the creation of hind limb ischemia in healthy rabbits through femoral artery ligation. In the past, however, there has been a strong disconnect between the success of preclinical studies and the failure of clinical trials regarding treatments for peripheral ischemia. Healthy animals typically have robust vascular regeneration in response to surgically induced ischemia, in contrast to the reduced vascularity and regeneration in patients with chronic peripheral ischemia. Here, we describe an optimized animal model for peripheral ischemia in rabbits that includes hyperlipidemia and diabetes. This model has reduced collateral formation and blood pressure recovery in comparison to a model with a higher cholesterol diet. Thus, the model may provide better correlation with human patients with compromised angiogenesis from the common co-morbidities that accompany peripheral vascular disease.

We describe a surgical procedure used to induce peripheral ischemia in rabbits with hyperlipidemia and diabetes. This surgery acts as a preclinical model for conditions experienced in peripheral artery disease in patients. Angiography is also described as a means to measure the extent of introduced ischemia and recovery of perfusion.

Modelo pré-clínico de isquemia de membros posteriores em coelhos diabéticos

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular ...