Este trabalho foi apoiado pela Key Project Medical Science and Technology Development Foundation, Nanjing Department of Health (No.YKK16098).

Os experimentos em animais foram aprovados pelo Comitê Institucional de Cuidados e Uso de Animais (IACUC) do Nanjing Drum Tower Hospital.
1. Preparação dos reagentes e animais
<ol><li>Dissolver o cloridrato de doxorubicina (Pfizer, EUA) em água esterilizada. Vórtice para obter uma solução Dox de 1 mg/mL e manter a 4 °C.</li>
	<li>Use camundongos C57BL/6 (8-10 semanas de idade; 25-30 g de peso). Para este estudo, os camundongos foram comprados no Centro de Pesquisa Animal Modelo da Universidade de Nanjing e mantidos na sala de animais do Nanjing Drum Tower Hospital.</li>
	<li>As gaiolas de camundongos livres de patógenos foram mantidas sob um ciclo claro/escuro de 12 h a uma temperatura constante de 23 °C. Todos os animais foram alimentados com uma dieta normal de comida e receberam comida e água ad libitum.</li>
</ol>2. Estabelecimento do modelo animal DCM
<ol><li>Randomize os camundongos em um grupo normal (n = 5) e dox (n = 5).</li>
	<li>Administre a solução Dox intraperitoneal a uma dose de 5 mg/kg usando uma seringa esterilizada de 1 mL 1x por semana para o grupo Dox. Trate os ratos de controle da mesma forma com a mesma quantidade de solução salina.</li>
	<li>Meça o peso corporal dos dois grupos semanalmente. Assim, ajuste a dose de injeção com base no peso corporal semanalmente por um total de 4 semanas, com uma dose acumulada de 20 mg/kg(Figura 1). NOTA: Um período de 4 semanas foi escolhido porque a ecocardiografia em 4 semanas mostrou diferença significativa na função cardíaca entre os dois grupos.</li>
</ol>3. Exame ecocardiográfico
<ol><li>No final da quarta semana, realize um exame ecocardiográfico dos camundongos.</li>
	<li>Anestesiar os camundongos com 2% de isoflurane intranasally. Se os camundongos não responderem a uma pitada da pele com uma pinça dentada ou estimular os dedos dos dedos e caudas, continue com o protocolo.</li>
	<li>Coloque o mouse em uma plataforma de manuseio de animais na posição supina. Para manter a anestesia, cubra o nariz e a boca do animal com um cone de nariz e entregue 2% de isoflurane. NOTA: Para anestesia IP, injete 4% de hidrato de cloro a uma dose de 0,2 mL/20 g.</li>
	<li>Remova a pele do peito cuidadosamente com uma máquina de barbear elétrica. Avalie a função cardíaca in vivo usando ecocardiografia transtorácica.</li>
	<li>Realize o ecocardiograma LV tanto nas visões parasternal de longo eixo quanto em eixo curto a uma taxa de quadros de 233 Hz. As dimensões sistólicas e terminais foram definidas como as fases correspondentes à onda ECG T e à onda R, respectivamente.</li>
	<li>Nos traçadores do modo M, meça o diâmetro médio lv end-sistólica (LVIDd), diâmetro diastólico final lv (LVIDs), espessura septal interventricular (IVS) e espessura posterior da parede LV (LVPW) de 3-5 batimentos cardíacos. Calcule também a fração de ejeção (EF) e o encurtamento de frações (FS) com base na ecocardiografia.</li>
</ol>4. Coloração histológica
<ol><li>Após a análise ecocardiográfica, sacrifique os camundongos por injeção intraventricular de 10% de cloreto de potássio.</li>
	<li>Perfunda o coração com cerca de 30 mL de soro fisiológico após dissecção até que o fígado e o pulmão fiquem pálidos.</li>
	<li>Extirpa o coração e lave-o completamente em solução tampão fosfato para extrusão sangue.</li>
	<li>Fixar o coração em 4% de formalina à temperatura ambiente por 24 h e tratar o tecido em uma caixa de parafina para que a cera de parafina esfrie e solidifique.</li>
	<li>Corte os corações em fatias de 5 μm de espessura para coloração patológica.</li>
	<li>De cera e seções de reidratação contendo músculo papilar.<ol>
<li>Incubar lâminas a 55 °C por 30 min. Em seguida, incubar em xileno 2x por 2 min cada; 100% etanol 2x por 2 min cada; 95% de etanol 2x por 2 min cada; 80% de etanol por 2 min; 75% de etanol por 2 min; e 50% de etanol por 2 min.</li>
	</ol>
</li>
	<li>Mancha usando hematoxilina e eosina (H&E), bem como a mancha de Masson.</li>
</ol>

<ol>
	<li>Weintraub, R. G., Semsarian, C., MacDonald, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dilated+cardiomyopathy.">Dilated cardiomyopathy.</a> Lancet. 390 (10092), 400-414 (2017).</li><li>Ichihara, S., 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=Attenuation+of+oxidative+stress+and+cardiac+dysfunction+by+bisoprolol+in+an+animal+model+of+dilated+cardiomyopathy.">Attenuation of oxidative stress and cardiac dysfunction by bisoprolol in an animal model of dilated cardiomyopathy.</a> Biochemical and Biophysical Research Communications. 350 (1), 105-113 (2006).</li><li>Fountoulakis, 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=Alterations+in+the+heart+mitochondrial+proteome+in+a+desmin+null+heart+failure+model.">Alterations in the heart mitochondrial proteome in a desmin null heart failure model.</a> Journal of Molecular and Cellular Cardiology. 38 (3), 461-474 (2005).</li><li>Fairweather, D., Rose, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coxsackievirus-induced+myocarditis+in+mice:+a+model+of+autoimmune+disease+for+studying+immunotoxicity.">Coxsackievirus-induced myocarditis in mice: a model of autoimmune disease for studying immunotoxicity.</a> Methods. 41 (1), 118-122 (2007).</li><li>Wang, Z. 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=A+therapeutic+anti-CD4+monoclonal+antibody+inhibits+T+cell+receptor+signal+transduction+in+mouse+autoimmune+cardiomyopathy.">A therapeutic anti-CD4 monoclonal antibody inhibits T cell receptor signal transduction in mouse autoimmune cardiomyopathy.</a> Chinese Medical Journal. 120 (15), 1319-1325 (2007).</li><li>Riad, A., 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=Toll-like+receptor-4+deficiency+attenuates+doxorubicin-induced+cardiomyopathy+in+mice.">Toll-like receptor-4 deficiency attenuates doxorubicin-induced cardiomyopathy in mice.</a> European Journal of Heart Failure. 10 (3), 233-243 (2008).</li><li>Kankeu, C., Clarke, K., Passante, E., Huber, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Doxorubicin-induced+chronic+dilated+cardiomyopathy-the+apoptosis+hypothesis+revisited.">Doxorubicin-induced chronic dilated cardiomyopathy-the apoptosis hypothesis revisited.</a> Journal of Molecular Medicine. 95 (3), 239-248 (2017).</li><li>Zhao, 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=MicroRNA-140-5p+aggravates+doxorubicin-induced+cardiotoxicity+by+promoting+myocardial+oxidative+stress+via+targeting+Nrf2+and+Sirt2.">MicroRNA-140-5p aggravates doxorubicin-induced cardiotoxicity by promoting myocardial oxidative stress via targeting Nrf2 and Sirt2.</a> Redox Biology. 15, 284-296 (2018).</li><li>O'Connell, J. 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=Short-term+and+long-term+models+of+doxorubicin-induced+cardiomyopathy+in+rats:+A+comparison+of+functional+and+histopathological+changes.">Short-term and long-term models of doxorubicin-induced cardiomyopathy in rats: A comparison of functional and histopathological changes.</a> Experimental and Toxicologic Pathology. 69 (4), 213-219 (2017).</li><li>Yuan, Y. P., 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=CTRP3+protected+against+doxorubicin-induced+cardiac+dysfunction,+inflammation+and+cell+death+via+activation+of+Sirt1.">CTRP3 protected against doxorubicin-induced cardiac dysfunction, inflammation and cell death via activation of Sirt1.</a> Journal of Molecular and Cellular Cardiology. 114, 38-47 (2018).</li><li>Sun, Z., 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+TGF-beta+pathway+mediates+doxorubicin+effects+on+cardiac+endothelial+cells.">The TGF-beta pathway mediates doxorubicin effects on cardiac endothelial cells.</a> Journal of Molecular and Cellular Cardiology. 90, 129-138 (2016).</li><li>Minotti, G., Menna, P., Salvatorelli, E., Cairo, G., Gianni, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anthracyclines:+molecular+advances+and+pharmacologic+developments+in+antitumor+activity+and+cardiotoxicity.">Anthracyclines: molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity.</a> Pharmacological Reviews. 56 (2), 185-229 (2004).</li><li>Vejpongsa, P., Yeh, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+of+anthracycline-induced+cardiotoxicity:+challenges+and+opportunities.">Prevention of anthracycline-induced cardiotoxicity: challenges and opportunities.</a> Journal of The American College of Cardiology. 64 (9), 938-945 (2014).</li><li>Renu, K., V, G. A., P, B. T., Arunachalam, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanism+of+doxorubicin-induced+cardiomyopathy+-+An+update.">Molecular mechanism of doxorubicin-induced cardiomyopathy - An update.</a> European Journal of Pharmacology. 818, 241-253 (2018).</li></ol>

Nenhum conflito de interesses declarado.

Dox é um medicamento de quimioterapia antitumor periódico inespecífico comumente utilizado na clínica12. Seu principal efeito colateral é a cardiotoxicidade, caracterizada por cardiomiopatia e subsequente insuficiência cardíaca13. O mecanismo subjacente inclui danos da peroxidação lipídica miocárdica, inibição do reticulum sarcoplasmático miocárdio Ca2+-ATIVIDADE ATPase, e ativação do sistema local de renin-angiotensina miocárdica, resultando em...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/61158">A Doxorubicin-Induced Murine Model of Dilated Cardiomyopathy In Vivo</a>. A figure was updated.
Figure 1 was updated from:
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61158/61158fig1v2.jpg" /> 
Figure 1: Schematic diagram of a Dox-induced dilated cardiomyopathy. <a href="https://www.jove.com/files/ftp_upload/61158/61158fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
to:
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61158/61158fig1v5.jpg" /> 
Figure 1: Schematic diagram of a Dox-induced dilated cardiomyopathy. <a href="https://www.jove.com/files/ftp_upload/61158/61158fig1v5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

An erratum was issued for:&#160;<a href="https://www.jove.com/t/61158">A Doxorubicin-Induced Murine Model of Dilated Cardiomyopathy In Vivo</a>. A figure was updated.

Erratum: A Doxorubicin-Induced Murine Model of Dilated Cardiomyopathy In Vivo

Uma das causas mais comuns de insuficiência cardíaca, o DCM é caracterizado pela dilatação ventricular e diminuição da função cardíaca e é a razão mais comum para transplante de coração em todo o mundo1. Para investigar melhor sua patogênese e encontrar tratamentos eficazes, o acesso a modelos animais maduros é especialmente importante. O objetivo dos experimentos descritos é estabelecer um modelo de rato estável de DCM que se assemelha ao DCM humano.
Devido à complexa patogênese do DCM, existem muitos métodos diferentes para fazer modelos animais correspondentes. Os modelos espontâneos de DCM2 são relativamente estáveis, mas são caros e não são facilmente disponíveis. Os modelos animais geneticamente modificados3 não são bem estabelecidos e requerem mais uso experimental. Modelos animais DCM induzidos por infecção viral4 ou defeitos autoimunes5 são fáceis de obter, mas não são totalmente representativos do DCM. Os modelos associados à toxicidade do miocárdio incluem modelos dcm induzidos pelo álcool e modelos animais DCM induzidos pelo Dox.
O modelo de cardiomiopatia induzido pelo Dox é obtido por injeção intraperitoneal de Dox6. O modelo explora o efeito colateral crônico mais grave do Dox: Após a exposição ao Dox, os pacientes desenvolvem sintomas de DCM de início tardio com uniformidade clínica7. O estresse oxidativo induzido pelo dox8 e o dano mitocondrial9, que levam à apoptose cardiomiocese, são sintomas na patogênese do DCM. Existem modelos de tratamento dox agudos e crônicos: uma única dose alta de Dox (15 mg/kg) induz um modelo de curto prazo para cardiomiopatia10, enquanto as injeções repetitivas de dox de baixa dose (seis semanais, 3 mg/kg) induzem um modelo de longo prazo para cardiomiopatia11. Com base no estudo apresentado, camundongos do tipo selvagem injetaram uma vez por semana durante um mês em uma dose de 5 mg/kg de morfologia e histologia do coração consistentes com as características do DCM ao final do tratamento, fornecendo uma maneira ideal de estabelecer um modelo de DCM.

<table>
	<tbody>
		<tr>
			<td>4% paraformaldehyde</td>
			<td>servicebio</td>
			<td>CAS30525-89-4</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6 mice</td>
			<td>Model Animal Research Center of Nanjing University</td>
			<td>\</td>
			<td></td>
		</tr>
		<tr>
			<td>Doxorubicin hydrochloride</td>
			<td>Pfizer</td>
			<td>CAS25316-40-9</td>
			<td></td>
		</tr>
		<tr>
			<td>echocardiography</td>
			<td>Visualsonics</td>
			<td>\</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin and Eosin staining kit</td>
			<td>Solarbio</td>
			<td>G1120</td>
			<td></td>
		</tr>
		<tr>
			<td>Masson staining kit</td>
			<td>Solarbio</td>
			<td>G1343</td>
			<td></td>
		</tr>
		<tr>
			<td>phosphate buffer solution</td>
			<td>Sigma</td>
			<td>P5368</td>
			<td></td>
		</tr>
		<tr>
			<td>potassium chloride</td>
			<td>Sigma</td>
			<td>CAS7447-40-7</td>
			<td></td>
		</tr>
		<tr>
			<td>sterilized syringe</td>
			<td>Millipore</td>
			<td>SLGP033RB</td>
			<td></td>
		</tr>
	</tbody>
</table>

a doxorubicin-induced murine model of dilated cardiomyopathy in vivo

A cardiomiopatia dilatada (DCM) refere-se a um espectro de distúrbios heterogêneos do miocárdio caracterizado por dilatação ventricular e desempenho cardíaco deprimido na ausência de hipertensão arterial, valvular, congênita ou doenças cardíacas isquêmicas, e que podem estar relacionadas a infecções, anormalidades autoimunes ou metabólicas ou herança familiar. Pode evoluir para insuficiência cardíaca congestiva com um prognóstico ruim. A doxorubicina (Dox) é amplamente empregada como uma droga quimioterápica, mas seu uso é limitado porque causa alterações semelhantes ao DCM do miocárdio. Sua toxicidade miocárdio é atribuída ao estresse oxidativo, inflamação crônica e apoptose cardiomiocócica. Um modelo de DCM explorando esses sintomas de DCM induzidos pelo Dox não foi estabelecido.

Função cardíaca A cardiomiopatia dilatada é caracterizada pela dilatação ventricular progressiva e disfunção contratil. A Figura 2 mostra imagens ecocardiográficas representativas dos dois grupos. Os camundongos tratados com dox apresentaram uma fração de ejeção ventricular esquerda significativamente reduzida e encurtamento fracionado ventricular esquerdo(Figura 3A,B). O diâmetro da LV também aumentou nas fase...

Watch this Scientific Journal Video about A Doxorubicin-Induced Murine Model of Dilated Cardiomyopathy In Vivo at JoVE.com

A Doxorubicin-Induced Murine Model of Dilated Cardiomyopathy In Vivo

Descrito é um protocolo para estabelecer um modelo de cardiomiopatia dilatada induzida por Doxorubicina (DCM) em camundongos através de injeção intraperitoneal de doxorubicina a longo prazo.

Um modelo de murina induzida por doxorubicina de cardiomiopatia dilatada em Vivo

Dilated cardiomyopathy (DCM) refers to a spectrum of heterogeneous myocardial disorders characterized by ventricular dilation and depressed cardiac ...

a-doxorubicin-induced-murine-model-of-dilated-cardiomyopathy-in-vivo

Research

JoVE Journal

Medicine

4.3K visualizações.  Clinical College of Nanjing Medical University. Descrito é um protocolo para estabelecer um modelo de cardiomiopatia dilatada induzida por Doxorubicina (DCM) em camundongos através de injeção intraperitoneal de doxorubicina a longo prazo.

Vídeo: A Doxorubicin-Induced Murine Model of Dilated Cardiomyopathy In Vivo

Development of Obliterative Bronchiolitis in a Murine Model of Orthotopic Lung Transplantation

Orthotopic lung transplantation in rats was first reported by Asimacopoulos and colleagues in 1971 1. Currently, this method is well accepted and standardized not only for the study of allo-rejection but also between syngeneic strains for examining mechanisms of ischemia-reperfusion injury after lung transplantation. Although the application of the rat and other large animal model 2 contributed significantly to the elucidation of these studies, the scope of those investigations is limited by the scarcity of knockout and transgenic rats. Due to no effective therapies for obliterative bronchiolitis, the leading cause of death in lung transplant patients, there has been an intensive search for pre-clinical models that replicate obliterative bronchiolitis. The tracheal allograft model is the most widely used and may reproduce some of the histopathologic features of obliterative bronchiolitis 3. However, the lack of an intact vasculature with no connection to the recipient's conducting airways, and incomplete pathologic features of obliterative bronchiolitis limit the utility of this model 4. Unlike transplantation of other solid organs, vascularized mouse lung transplants have only recently been reported by Okazaki and colleagues for the first time in 2007 5. Applying the basic principles of the rat lung transplant, our lab initiated the obliterative bronchiolitis model using minor histoincompatible antigen murine orthotopic single-left lung transplants which allows the further study of obliterative bronchiolitis immunopathogenesis6.

Obliterative bronchiolitis is the key impediment to the long-term survival of lung transplant recipients and the lack of a robust preclinical model precludes examining obliterative bronchiolitis immunopathogenesis. Unlike other solid organ transplants, vascularized mouse lung transplantation has only recently been developed. Here we show our independently developed obliterative bronchiolitis model after murine orthotopic single-lung transplantation.

Desenvolvimento de bronquiolite obliterante em um modelo murino de transplante de pulmão ortotópico

Orthotopic lung transplantation in rats was first reported by Asimacopoulos and colleagues in 1971 1. Currently, this method is well accepted and ...

Medicina

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 ...

A Preclinical Murine Model of Hepatic Metastases

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, murine pancreatic tumor model of hepatic metastases via a hemispleen injection of syngeneic murine pancreatic tumor cells is described. This model mimics many of the clinical conditions in patients with metastatic disease to the liver. Mice consistently develop metastases in the liver allowing for investigation of the metastatic process, experimental therapy testing, and tumor immunology research.

A preclinical, murine model of hepatic metastases performed via a hemispleen injection technique.

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, ...

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

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

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

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

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

Tachycardia-Induced Cardiomyopathy As a Chronic Heart Failure Model in Swine

A stable and reliable model of chronic heart failure is required for many experiments to understand hemodynamics or to test effects of new treatment methods. Here, we present such a model by tachycardia-induced cardiomyopathy, which can be produced by rapid cardiac pacing in swine.
A single pacing lead is introduced transvenously into fully anaesthetized healthy swine, to the apex of the right ventricle, and fixated. Its other end is then tunneled dorsally to the paravertebral region. There, it is connected to an in-house modified heart pacemaker unit that is then implanted in a subcutaneous pocket. 
After 4 - 8 weeks of rapid ventricular pacing at rates of 200 - 240 beats/min, physical examination revealed signs of severe heart failure - tachypnea, spontaneous sinus tachycardia, and fatigue. Echocardiography and X-ray showed dilation of all heart chambers, effusions, and severe systolic dysfunction. These findings correspond well to decompensated dilated cardiomyopathy and are also preserved after the cessation of pacing.
This model of tachycardia-induced cardiomyopathy can be used for studying the pathophysiology of progressive chronic heart failure, especially hemodynamic changes caused by new treatment modalities like mechanical circulatory supports. This methodology is easy to perform and the results are robust and reproducible.

Here, we present a protocol to produce tachycardia-induced cardiomyopathy in swine. This model represents a potent way to study the hemodynamics of progressive chronic heart failure and the effects of applied treatment.

Cardiomiopatia induzida por taquicardia como um modelo de insuficiência cardíaca crônica em suínos

A stable and reliable model of chronic heart failure is required for many experiments to understand hemodynamics or to test effects of new treatment ...

A Doxorubicin-induced Cardiomyopathy Model in Adult Zebrafish

The genetically accessible adult zebrafish (Danio rerio) has been increasingly used as a vertebrate model for understanding human diseases such as cardiomyopathy. Because of its convenience and amenability to high throughput genetic manipulations, the generation of acquired cardiomyopathy models, such as the doxorubicin-induced cardiomyopathy (DIC) model in adult zebrafish, is opening the doors to new research avenues, including discovering cardiomyopathy modifiers via forward genetic screening. Different from the embryonic zebrafish DIC model, both initial acute and later chronic phases of cardiomyopathy can be determined in the adult zebrafish DIC model, enabling the study of stage-dependent signaling mechanisms and therapeutic strategies. However, variable results can be obtained with the current model, even in the hands of experienced investigators. To facilitate future implementation of the DIC model, we present a detailed protocol on how to generate this DIC model in adult zebrafish and describe two alternative ways of intraperitoneal (IP) injection. We further discuss options on how to reduce variations to obtain reliable results and provide suggestions on how to appropriately interpret the results.

A method to generate a doxorubicin-induced cardiomyopathy model in adult zebrafish (Danio rerio) is described here. Two alternative ways of intraperitoneal injection are presented and conditions to reduce variations among different experimental groups are discussed.

Um modelo de cardiomiopatia induzida por doxorrubicina no Zebrafish adulto

The genetically accessible adult zebrafish (Danio rerio) has been increasingly used as a vertebrate model for understanding human diseases such as ...

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 ...

Assessing Therapeutic Angiogenesis in a Murine Model of Hindlimb Ischemia

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard therapy, a considerable number of CLI patients are not suited for either surgical or endovascular revascularization. Angiogenic therapies are emerging as an option for these patients but are currently still under investigation. Before application in humans, those therapies must be tested in animal models and its mechanisms must be clearly understood. An animal model of hindlimb ischemia (HLI) has been developed by the ligation and excision of the distal external iliac and femoral arteries and veins in mice. A comprehensive panel of tests was assembled to assess the effects of ischemia and putative angiogenic therapies at functional, histologic and molecular levels. Laser Doppler was used for the flow measurement and functional assessment of perfusion. Tissue response was evaluated by the analysis of capillary density after staining with the anti-CD31 antibody on histological sections of gastrocnemius muscle and by measurement of collateral vessel density after diaphonization. Expression of angiogenic genes was quantified by RT-PCR targeting selected angiogenic factors exclusively in endothelial cells (ECs) after laser capture microdissection from mice gastrocnemius muscles. These methods were sensitive in identifying differences between ischemic and non-ischemic limbs and between treated and non-treated limbs. This protocol provides a reproducible model of CLI and a framework for testing angiogenic therapies.

Here, a critical hindlimb ischemia experimental model is presented followed by a battery of functional, histologic and molecular tests&#160;to assess the effectiveness of angiogenic therapies.&#8203;

Avaliando a angiogênese terapêutica em um modelo murino de isquemia Retromembro

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard ...

In Vivo Mouse Model of Spinal Implant Infection

Spine implant infections portend poor outcomes as diagnosis is challenging and surgical eradication is at odds with mechanical spinal stability. The purpose of this method is to describe a novel mouse model of spinal implant infection (SII) that was created to provide an inexpensive, rapid, and accurate in vivo tool to test potential therapeutics and treatment strategies for spinal implant infections.
In this method, we present a model of posterior-approach spinal surgery in which a stainless-steel k-wire is transfixed into the L4 spinous process of 12-week old C57BL/6J wild-type mice and inoculated with 1 x 103 CFU of a bioluminescent strain of Staphylococcus aureus Xen36 bacteria. Mice are then longitudinally imaged for bioluminescence in vivo on post-operative days 0, 1, 3, 5, 7, 10, 14, 18, 21, 25, 28, and 35. Bioluminescence imaging (BLI) signals from a standardized field of view are quantified to measure in vivo bacterial burden.
To quantify bacteria adhering to implants and peri-implant tissue, mice are euthanized and the implant and surrounding soft tissue are harvested. Bacteria are detached from the implant by sonication, cultured overnight and then colony forming units (CFUs) are counted. The results acquired from this method include longitudinal bacterial counts as measured by in vivo S. aureus bioluminescence (mean maximum flux) and CFU counts following euthanasia.
While prior animal models of instrumented spine infection have involved invasive, ex vivo tissue analysis, the mouse model of SII presented in this paper leverages noninvasive, real time in vivo optical imaging of bioluminescent bacteria to replace static tissue study. Applications of the model are broad and may include utilizing alternative bioluminescent bacterial strains, incorporating other types of genetically engineered mice to contemporaneously study host immune response, and evaluating current or investigating new diagnostic and therapeutic modalities such as antibiotics or implant coatings.

The protocol describes a novel in vivo mouse model of spinal implant infection where a stainless-steel k-wire implant is infected with bioluminescent Staphylococcus aureus Xen36. Bacterial burden is monitored longitudinally with bioluminescent imaging and confirmed with colony forming unit counts after euthanasia.

Modelo in vivo de infecção de implante espinhal em camundongos

Spine implant infections portend poor outcomes as diagnosis is challenging and surgical eradication is at odds with mechanical spinal stability. The ...