XHTW é um WM Keck Foundation Scholar Distinguished Jovens em Pesquisa Médica, e também é suportado pelo NIH / NHLBI concede R01-R01-HL089598 e R01HL091947 e Muscular Dystrophy Association conceder # 69238. RJvO é o destinatário da Fellowship 2008-2010 American Physiological Society Pós-Doutorado em Physiological Genomics. Este trabalho também é suportado, em parte, pela Aliança para a Fondation Leducq CaMKII Sinalização no Coração.

Parte 1: Preparação do campo operatório <ol><li> O campo de operação é desinfetada com 75% de álcool isopropílico. </li><li> Certifique-se a almofada de aquecimento está ligado e na temperatura certa. Um sistema recomendado é uma bomba de água Gaymar circulando ligada a um bloco de terapia que é mantida a 37 ° C ± 1 ° C. É importante para manter a temperatura corporal normal durante a cirurgia, para evitar uma rápida diminuição da freqüência cardíaca. </li><li> Instrumentos cirúrgicos são esterilizados em estufa quente talão antes da cirurgia. Para este procedimento, você vai precisar dos seguintes instrumentos cirúrgicos: uma tesoura sem corte, é claro curvas forceps X 2, multa de 45 ° pinças anguladas X 2, tesoura primavera angulado, afastador peito, e porta-agulha. </li><li> Aplicadores de algodão deve estar na mão em caso de hemorragia. </li></ol> Parte 2: Preparação e intubação de Ratos <ol><li> Os ratos são anestesiados em uma câmara de indução com isoflurano a 2% misturado com 0,5 -1,0 L / min 100% O 2. </li><li> Cortar cabelo são usados ​​para raspar a pele do decote no peito ao nível médio. </li><li> O mouse é colocado em posição supina sobre uma almofada de aquecimento, a fim de manter a temperatura corporal. </li><li> A faixa de borracha é colocada sobre dentes do animal s da frente para estender o pescoço. Utilizando uma pinça curva em uma das mãos, a língua é delicadamente manipulado para o lado. Com a outra mão, a intubação traqueal é realizada utilizando tubos PE 90. O tubo endotraqueal é então ligado a um ventilador Harvard ciclismo volumétricos de roedores em 125-150 respirações / minuto e um volume corrente de 0,1-0,3 ml. Durante o procedimento cirúrgico, a anestesia é mantida em 1,5-2% com isoflurano 0,5-1,0 L / min 100% O 2. Nível correto de anestesia é verificada através da aplicação de pressão sobre o leito ungueal do mouse (tep pinch-reflex). </li><li> O campo cirúrgico é desinfetada com uma solução de betadine seguido pelo álcool 70%. Este procedimento é repetido três vezes. </li><li> Para evitar a contaminação do campo cirúrgico durante a operação, uma cortina estéril é colocado sobre o mouse, deixando apenas o campo de operação exposta. </li><li> Um conjunto de luvas estéreis é utilizada para cada rato individual. </li></ol> Parte 3: A ligadura da aorta transversa <ol><li> Toracotomia parcial para a segunda costela é realizada sob um microscópio cirúrgico eo esterno retraído com afastador peito. </li><li> Ponta fina 45 ° pinças anguladas são usados ​​para separar delicadamente o tecido do timo e gordura do arco aórtico. </li><li> Após a identificação da aorta transversa, um pequeno pedaço de um fio de seda 6.0 é colocado entre as artérias inominada e carótida esquerda (Figura 1). </li><li> Dois nós soltas são amarradas ao redor da aorta transversa e um pequeno pedaço de uma agulha de 27 ½ blunt indicador é colocado paralelamente à aorta transversa. O primeiro nó é rapidamente amarrado contra a agulha, seguido pelo segundo e da agulha prontamente removidos a fim de produzir uma constrição de 0,4 milímetros de diâmetro. Em camundongos do grupo controle, todo o procedimento é idêntico, exceto para a ligadura da aorta. </li><li> O afastador de tórax é removido e os de saída do ventilador pinched off por 2 segundos para re-inflar os pulmões. </li><li> A caixa torácica é fechado com uma sutura prolene 6.0 com um padrão de sutura interrompido. </li><li> A pele é fechada com uma sutura prolene 6.0 com um padrão de sutura contínua. </li></ol> Parte 4: recuperação pós-operatória <ol><li> Para analgesia pós-operatória, o mouse é injetado com buprenorfina (0,1 mg / kg) por via intraperitoneal. Se houver sinais de desidratação após a cirurgia, solução salina estéril é dada por via intraperitoneal. </li><li> A anestesia é gradualmente reduzido para a posição off e do tubo endotraqueal removido quando os sinais de respiração espontânea ocorrer. </li><li> O mouse é movido para a posição prona e permitiu recuperar em uma almofada de aquecimento. </li></ol> Parte 5: Confirmação de ligadura de sucesso da aorta transversa <ol><li> Uma semana após a TAC, o mouse é re-anestesiados para determinar o grau de sobrecarga de pressão induzida por ligadura da aorta transversa. Como descrito acima, a anestesia é mantida em 1,5-2% com isoflurano 0,5-1,0 L / min 100% O 2 e temperatura corporal mantida a 37 ° C ± 1 ° C. </li><li> A 20 MHz Doppler sonda é colocada sobre os lados esquerdo e direito do pescoço em um ângulo de 45 ° para detectar velocidades de fluxo. Um computador baseado em processador de sinal Doppler (Indus Instruments, Houston, TX) foi utilizado para exibir e armazenar sinais de Doppler 5. </li><li> Dependendo do grau de sobrecarga de pressão necessária para o protocolo experimental, os ratos apenas com um carótida direita (RC) / carótida esquerda (LC) relação de fluxo em um determinado intervalo estão incluídos para análise posterior. Por exemplo, um grau moderado de sobrecarga de pressão leva a uma proporção de 5-8, enquanto que um apertado constrição resultando em sobrecarga de pressão leva grave toa proporção de 8-10 (Figura 5.3A). Um animal sham (operado, mas não ligada), no entanto, espera-se ter uma proporção de aproximadamente 1 (Figura 5.3b). </li></ol> 6: Resultados Representante A sobrevivência típica de intervenção cirúrgica após TAC em camundongos selvagens é de cerca de 80-90%. Ligadura cirúrgica bem sucedida da aorta transversa levará a uma relação de velocidade de fluxo Doppler entre direita e esquerda da artéria carótida (RC / LC) de 5-10 (Figura 1). Comparado com o sham-operado camundongos (Figura 2A), ratos com sobrecarga de pressão devem desenvolver hipertrofia cardíaca dentro de 1-2 semanas, e dilatação cardíaca após 6-8 semanas, dependendo da tensão da constrição (Figura 2B). Mortalidade durante o primeiro mês após o TAC é tipicamente baixa (&lt;20%) em camundongos selvagens, apesar de camundongos geneticamente modificados podem apresentar diferentes taxas de sobrevivência 6,7. <img src="/files/ftp_upload/1729/1729fig1tmb.jpg" alt="Figura 1" border="0" /> Figura 1. Confirmação da Ligadura aorta transversa com Doppler medição de velocidade. Representante sinais de velocidade Doppler do direito (RC) e carótida esquerda (LC) artérias seis dias pós-TAC. Essas gravações demonstram uma ligadura de sucesso com uma taxa de fluxo de RC / LC de ~ 6.8. Por favor, <a href="https://www.jove.com/files/ftp_upload/1729/1729fig1.jpg">clique aqui</a> para ver uma versão ampliada da figura 1. <img src="/files/ftp_upload/1729/1729fig2.jpg" alt="Figura 2" /> Figura 2. Representante Whole Imagens Coração Após TAC. A B. Sham operados coração de rato tipo selvagem.. Coração de rato tipo selvagem 16 semanas pós-TAC. Cada linha = 1 milímetro.

<ol>
	<li>Rockman, H. A., Ross, R. S., Harris, A. N., Knowlton, K. U., Steinhelper, M. E., Field, L. J., Ross, J., Chein, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segregation+of+atrial-specific+and+inducible+expression+of+an+atrial+natriuretic+factor+transgene+in+an+in+vivo+murine+model+of+cardiac+hypertrophy.">Segregation of atrial-specific and inducible expression of an atrial natriuretic factor transgene in an in vivo murine model of cardiac hypertrophy.</a> Proc. Natl. Acad. Sci. USA. 88, 8277-8281 (1991).</li><li>Heineke, J., Molkentin, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+cardiac+hypertrophy+by+intracellular+signaling+pathways.">Regulation of cardiac hypertrophy by intracellular signaling pathways.</a> Nat. Rev. Mol. Cell. Biol. 7, 589-600 (2006).</li><li>Hartley, C. J., Reddy, A. K., Madala, S., Michael, L. H., Entman, M. L., Taffett, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Doppler+estimation+of+reduced+coronary+flow+reserve+in+mice+with+pressure+overload+cardiac+hypertrophy.">Doppler estimation of reduced coronary flow reserve in mice with pressure overload cardiac hypertrophy.</a> Ultrasound Med. Biol. 34, 892-901 (2008).</li><li>Reddy, A. K., Taffett, G. E., Li, Y. -. H., Lim, S. -. W., Pham, T. T., Pocius, J. S., Entman, M. L., Michael, L. H., Hartley, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Doppler+signal+processing+for+use+in+mice:+applications.">Doppler signal processing for use in mice: applications.</a> IEEE Trans. Biomed. Eng. 52, 1771-1783 (2005).</li><li>Li, Y. -. H., Reddy, A. K., Ochoa, L. N., Pham, T. T., Hartley, C. J., Micheal, L. H., Entman, M. L., Taffett, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+age+on+peripheral+vascular+response+to+transverse+aortic+banding+in+mice.">Effect of age on peripheral vascular response to transverse aortic banding in mice.</a> J. Gerontol. 58A, 895-899 (2003).</li><li>Bourajjaj, M., Armand, A. S., Martins, d. a. C. o. s. t. a., Weijts, P. A., Nagel, B. V. a. n. d. e. r., Heeneman, R., Wehrens, S., Windt, X. H. D. e., J, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NFATc2+is+a+necessary+mediator+of+calcineurin-dependent+cardiac+hypertrophy+and+heart+failure.">NFATc2 is a necessary mediator of calcineurin-dependent cardiac hypertrophy and heart failure.</a> J. Biol. Chem. 28, 22295-22303 (2008).</li><li>Oort, R. J. v. a. n., Respress, J. L., Li, N., Reynolds, C., Almeida, A. C. D. e., Skapura, D. G., Windt, L. J. D. e., Wehrens, X. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+development+of+pressure+overload+induced+cardiac+hypertrophy+and+dysfunction+in+an+RyR2-R176Q+knockin+mouse+model.">Accelerated development of pressure overload induced cardiac hypertrophy and dysfunction in an RyR2-R176Q knockin mouse model.</a> Hypertension. 55, (2010).</li><li>Tarnavski, O., McMullen, J. R., Schinke, M., Nie, Q., Kong, S., Izumo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+cardiac+surgery:+comprehensive+techniques+for+the+generation+of+mouse+models+of+human+diseases+and+their+application+for+genomic+studies.">Mouse cardiac surgery: comprehensive techniques for the generation of mouse models of human diseases and their application for genomic studies.</a> Physiol Genomics. 16, 349-360 (2004).</li></ol>

TAC, que imita estenose aórtica humana, é um método comum para induzir hipertrofia cardíaca e insuficiência cardíaca em camundongos. Locais alternativos para a constrição da aorta incluem a aorta ascendente e abdominal. Ascendente constrição aórtica fornece uma sobrecarga extrema e mais rápida sobre o ventrículo esquerdo (LV). Em contraste, a constrição da aorta abdominal deixa intacta uma porção maior da circulação como meio de compensação possível. 8 Portanto, TAC é muitas vezes o mod...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Gaymar T/pump</td>
<td>&nbsp;</td>
<td>Harvard Apparatus</td>
<td>Model TP-500</td>
<td>Circulating water pump</td>
</tr>
<tr>
<td>Temp. therapy pads</td>
<td>&nbsp;</td>
<td>Harvard Apparatus</td>
<td>60-3414</td>
<td>&nbsp;</td>
<tr>
<td>Hot bead sterilizer</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>18000-45</td>
<td>&nbsp;</td>
<tr>
<td>Blunt scissors</td>
<td>&nbsp;</td>
<td>Roboz</td>
<td>RS-5980</td>
<td>&nbsp;</td>
<tr>
<td>Angled spring scissors</td>
<td>&nbsp;</td>
<td>Roboz</td>
<td>RS-5668</td>
<td>&nbsp;</td>
<tr>
<td>Chest retractor</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>17002-02</td>
<td>&nbsp;</td>
<tr>
<td>Course curved forceps</td>
<td>&nbsp;</td>
<td>Roboz</td>
<td>RS-5138</td>
<td>&nbsp;</td>
<tr>
<td>Dumont fine 45&deg; angled forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11253-25</td>
<td>&nbsp;</td>
<tr>
<td>Needle holder</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>12565-14</td>
<td>&nbsp;</td>
<tr>
<td>Electric shaver</td>
<td>&nbsp;</td>
<td>General Electric</td>
<td>&nbsp;</td>
<td>For shaving mouse fur</td>
</tr>
<tr>
<td>PE 90 tubing</td>
<td>&nbsp;</td>
<td>BD</td>
<td>427420</td>
<td>For intubation</td>
</tr>
<tr>
<td>MiniVent (ventilator)</td>
<td>&nbsp;</td>
<td>Harvard Apparatus</td>
<td>Type 845</td>
<td>&nbsp;</td>
<tr>
<td>Betadine</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>19-027132</td>
<td>May be purchased at medical supply store</td>
</tr>
<tr>
<td>Sterile gloves</td>
<td>&nbsp;</td>
<td>Mckesson</td>
<td>20-1565</td>
<td>&nbsp;</td>
<tr>
<td>Stereo microscope</td>
<td>&nbsp;</td>
<td>Unico</td>
<td>ZM186</td>
<td>&nbsp;</td>
<tr>
<td>6-0 silk suture</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>18020-60</td>
<td>&nbsp;</td>
<tr>
<td>27 &frac12; gauge needle</td>
<td>&nbsp;</td>
<td>BD</td>
<td>305109</td>
<td>&nbsp;</td>
<tr>
<td>6-0 prolene suture</td>
<td>&nbsp;</td>
<td>Mckesson</td>
<td>3286</td>
<td>&nbsp;</td>
<tr>
<td>Buprenorphine</td>
<td>&nbsp;</td>
<td>Institutional Animal Facility</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
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		</div>

transverse aortic constriction in mice

Constrição aórtica transversal (TAC) no rato é um modelo experimental comumente usado para a pressão de sobrecarga induzida hipertrofia cardíaca e insuficiência cardíaca.<sup&gt; 1</sup&gt; TAC inicialmente leva a hipertrofia compensada do coração, que muitas vezes está associada a um aumento temporário da contratilidade cardíaca. Ao longo do tempo, no entanto, a resposta à sobrecarga hemodinâmica crônica torna-se maladaptive, resultando em dilatação cardíaca e insuficiência cardíaca.<sup&gt; 2</sup&gt; O modelo murino TAC foi validado por Rockman<em&gt; Et al</em&gt;.<sup&gt; 1</sup&gt;, E desde então tem sido amplamente utilizado como uma ferramenta valiosa para imitar humanos doenças cardiovasculares e elucidar fundamentais processos de sinalização envolvidos na resposta hipertrófica cardíaca e desenvolvimento de insuficiência cardíaca. Quando comparado a outros modelos experimentais de insuficiência cardíaca, tais como oclusão completa da descendente anterior esquerda (DAE) da artéria coronária, TAC fornece um modelo mais reprodutível de hipertrofia cardíaca e um curso de tempo mais gradual no desenvolvimento de insuficiência cardíaca. Aqui, descrevemos um procedimento passo-a-passo para realizar TAC cirúrgica em camundongos. Para determinar o nível de sobrecarga de pressão produzida pela ligadura da aorta, uma sonda de alta freqüência Doppler é usado para medir a relação entre velocidades de fluxo sanguíneo nas artérias carótidas direita e esquerda.<sup&gt; 3, 4</sup&gt; Com as taxas de sobrevivência cirúrgica de 80-90%, aórtico transverso bandagem é uma técnica eficaz de induzir a hipertrofia ventricular esquerda e insuficiência cardíaca em camundongos.

Watch this Scientific Journal Video about Transverse Aortic Constriction in Mice at JoVE.com

Transverse Aortic Constriction in Mice

Constrição aórtica transversal (TAC) no rato é um modelo experimental comumente usado para estudar os mecanismos subjacentes a hipertrofia cardíaca eo desenvolvimento de insuficiência cardíaca. Aqui, descrevemos os procedimentos a constrição da aorta para criar um grau reprodutível de hipertrofia cardíaca em camundongos.

Constrição aórtica transversais em ratos

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model for pressure overload-induced cardiac hypertrophy and heart ...

Research

JoVE Journal

Biology

66.7K visualizações.  Baylor College of Medicine (BCM). Constrição aórtica transversal (TAC) no rato é um modelo experimental comumente usado para estudar os mecanismos subjacentes a hipertrofia cardíaca eo desenvolvimento de insuficiência cardíaca. Aqui, descrevemos os procedimentos a constrição da aorta para criar um grau reprodutível de hipertrofia cardíaca em camundongos.

Vídeo: Transverse Aortic Constriction in Mice

Small Bowel Transplantation In Mice

Since 1990, the development of tacrolimus-based immunosuppression and improved surgical techniques, the increased array of potent immunosuppressive medications, infection prophylaxis, and suitable patient selection helped improve actuarial graft and patient survival rates for all types of intestine transplantation. Patients with irreversible intestinal failure and complications of parenteral nutrition should now be routinely considered for small intestine transplantation. However, Survival rates for small intestinal transplantation have been slow to improve compares increasingly favorably with renal, liver, heart and lung. The small bowel transplantation is still unsatisfactory compared with other organs. Further progress may depend on better understanding of immunology and physiology of the graft and can be greatly facilitated by animal models. A wider use of mouse small bowel transplantation model is needed in the study of immunology and physiology of the transplantation gut as well as efficient methods in diagnosing early rejection. However, this model is limited to use because the techniques involved is an extremely technically challenging. We have developed a modified technique. When making anastomosis of portal vein and inferior vena cava, two stay sutures are made at the proximal apex and distal apex of the recipient s inferior vena cava with the donor s portal vein. The left wall of the inferior vena cava and donor s portal vein is closed with continuing sutures in the inside of the inferior vena cava after, after one knot with the proximal apex stay suture the right wall of the inferior vena cava and the donor s portal vein are closed with continuing sutures outside the inferior vena cave with 10-0 sutures. This method is easier to perform because anastomosis is made just on the one side of the inferior vena cava and 10-0 sutures is the right size to avoid bleeding and thrombosis.  In this article, we provide details of the technique to supplement the video.

The mouse small bowel transplantation model has been recognized as an important tool to study mechanismes of immune rejection and screen new immunosuppressive drugs. However, this model is limited to use because the techniques involved is an extremely technically challenge. Now we introduce the modified technique.

Transplante de intestino delgado em ratos

Since 1990, the development of tacrolimus-based immunosuppression and improved surgical techniques, the increased array of potent immunosuppressive ...

Biologia

Aortic Ring Assay

Angiogenesis, the sprouting of blood vessels from preexisting vasculature is associated with both natural and pathological processes. Various angiogenesis assays involve the study of individual endothelial cells in culture conditions (1). The aortic ring assay is an angiogenesis model that is based on organ culture. In this assay, angiogenic vessels grow from a segment of the aorta (modified from (2)). Briefly, mouse thoracic aorta is excised, the fat layer and adventitia are removed, and rings approximately 1 mm in length are prepared. Individual rings are then embedded in a small solid dome of basement matrix extract (BME), cast inside individual wells of a 48-well plate. Angiogenic factors and inhibitors of angiogenesis can be directly added to the rings, and a mixed co-culture of aortic rings and other cell types can be employed for the study of paracrine angiogenic effects. Sprouting is observed by inspection under a stereomicroscope over a period of 6-12 days. Due to the large variation caused by the irregularities in the aortic segments, experimentation in 6-plicates is strongly advised. Neovessel outgrowth is monitored throughout the experiment and imaged using phase microscopy, and supernatants are collected for measurement of relevant angiogenic and anti-angiogenic factors, cell death markers and nitrite.

Angiogenesis, the sprouting of blood vessels from pre-existing vasculature, is associated with both natural and pathological processes. Here we demonstrate an aortic ring assay that allows angiogenic potentiators and inhibitors to be directly added to aortic rings in culture. Sprouting and neovessel outgrowth can be determined by inspecting the aortic rings over a period of 6-12 days.

Aórtica Assay Anel

Angiogenesis, the sprouting of blood vessels from preexisting vasculature is associated with both natural and pathological processes. Various angiogenesis ...

Ambulatory ECG Recording in Mice

Telemetric ECG recording in mice is essential to understanding the mechanisms behind arrhythmias, conduction disorders, and sudden cardiac death. Although the surface ECG is utilized for short-term measurements of waveform intervals, it is not practical for long-term studies of heart rate variability or the capture of rare episodes of arrhythmias. Implantable ECG telemeters offer the advantages of simple surgical implantation, long-term recording of electrograms in ambulatory mice, and scalability with simultaneous recordings of multiple animals. Here, we present a step-by-step guide to the implantation of telemeters for ambulatory ECG recording in mice. Careful adherence to aseptic technique is required for favorable survival results with the possibility of implantation and recording from weeks to months. Thus, implantable ECG telemetry is a valuable tool for detection of critical information on cardiac electrophysiology in ambulatory animal models such as the mouse. 

Telemetric ECG has emerged as an essential tool in evaluating animal models for cardiac arrhythmias and sudden cardiac death. Here, we present a stepwise guide to telemetric ECG recordings for application in long-term ambulatory ECG monitoring in mice.

Gravação de ECG ambulatorial em Ratos

Telemetric ECG recording in mice is essential to understanding the mechanisms behind arrhythmias, conduction disorders, and sudden cardiac death. Although ...

Intravenous Injections in Neonatal Mice

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are technically feasible and routine. However, some mouse models can have early onset of disease with a rapid progression that makes administration of potential therapies difficult. The temporal (or facial) vein is just anterior to the ear bud in mice and is clearly visible for the first two days after birth on either side of the head using a dissecting microscope. During this window, the temporal vein can be injected with volumes up to 50 &#956;l. The injection is safe and well tolerated by both the pups and the dams. A typical injection procedure is completed within 1-2 min, after which the pup is returned to the home cage. By the third postnatal day the vein is difficult to visualize and the injection procedure becomes technically unreliable. This technique has been used for delivery of adeno-associated virus (AAV) vectors, which in turn can provide almost body-wide, stable transgene expression for the life of the animal depending on the viral serotype chosen.

Animal models of pediatric disease can experience early onset and aggressive disease progression. Clinically relevant therapy delivery to young mouse models can be technically difficult. This protocol describes a non-invasive intravenous injection method for newborn mice within the first two postnatal days of life.

Injeções intravenosas em Neonatal Ratos

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

Isolamento e cultura primária de células de camundongo aórtica endoteliais

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Induction of Right Ventricular Failure by Pulmonary Artery Constriction and Evaluation of Right Ventricular Function in Mice

The mechanism of right ventricular failure (RVF) requires clarification due to the uniqueness, high morbidity, high mortality, and refractory nature of RVF. Previous rat models imitating RVF progression have been described. Compared with rats, mice are more accessible, economical, and widely used in animal experiments. We developed a pulmonary artery constriction (PAC) approach which is comprised of banding the pulmonary trunk in mice to induce right ventricular (RV) hypertrophy. A special surgical latch needle was designed that allows for easier separation of the aorta and the pulmonary trunk. In our experiments, the use of this fabricated latch needle reduced the risk of arteriorrhexis and improved the surgical success rate to 90%. We used different padding needle diameters to precisely create quantitative constriction, which can induce different degrees of RV hypertrophy. We quantified the degree of constriction by evaluating the blood flow velocity of the PA, which was measured by noninvasive transthoracic echocardiography. RV function was precisely evaluated by right heart catheterization at 8 weeks after surgery. The surgical instruments made inhouse were composed of common materials using a simple process that is easy to master. Therefore, the PAC approach described here is easy to imitate using instruments made in the lab and can be widely used in other labs. This study presents a modified PAC approach that has a higher success rate than other models and an 8-week postsurgery survival rate of 97.8%. This PAC approach provides a useful technique for studying the mechanism of RVF and will enable an increased understanding of RVF.

Here, we provide a useful approach for studying the mechanism of right ventricular failure. A more convenient and efficient approach to pulmonary artery constriction is established using surgical instruments made inhouse. In addition, methods to evaluate the quality of this approach by echocardiography and catheterization are provided.

Indução de insuficiência ventricular direita por constrição da artéria pulmonar e avaliação da função ventricular direita em camundongos

The mechanism of right ventricular failure (RVF) requires clarification due to the uniqueness, high morbidity, high mortality, and refractory nature of ...

Inducing Apical Periodontitis in Mice

The mechanisms involved in local induced inflammation can be studied using several available animal models. One of these is the induction of apical periodontitis (AP). Apical periodontitis is a common pathology of an inflammatory nature in the periodontal tissues surrounding the tooth root. In order to better understand the nature and mechanism of this pathology it is advantageous to perform the procedure in mice. The induction of this odontogenic inflammation is achieved by drilling into the mouse tooth until the dental pulp is exposed. Next, the tooth pulp remains exposed to be contaminated by the natural oral flora over time, causing apical periodontitis. After this time period, the animal is sacrificed, and the tooth and the jaw bone can be analyzed in various ways. Typical analyses include micro-CT imaging (to evaluate bone resorption), histological staining, immunohistochemistry, and RNA expression. This protocol is useful for research in the field of oral biology to better understand this inflammatory process in an in vivo experimental setting with uniform conditions. The procedure requires a careful handling of the mice and the isolated jaw, and a visual demonstration of the technique is useful. All technical aspects of the procedures leading to induced apical periodontitis and its characterization in a mouse model are demonstrated.

Here, we present a protocol to locally induce apical periodontitis in mice. We show how to drill a hole in the mouse&#39;s tooth and expose its pulp, in order to cause local inflammation. Analysis methods to investigate the nature of this inflammation, such as micro-CT and histology, are also demonstrated.

Induzindo a periodontite apical em camundongos

The mechanisms involved in local induced inflammation can be studied using several available animal models. One of these is the induction of apical ...

Transverse Fracture of the Mouse Femur with Stabilizing Pin

Fracture repair is an essential function of the skeleton that cannot be reliably modeled in vitro. A mouse injury model is an efficient approach to test whether a gene, gene product or drug influences bone repair because murine bones recapitulate the stages observed during human fracture healing. When a mouse or human breaks a bone, an inflammatory response is initiated, and the periosteum, a stem cell niche surrounding the bone itself, is activated and expands. Cells residing in the periosteum then differentiate to form a vascularized soft callus. The transition from the soft callus to a hard callus occurs as the recruited skeletal progenitor cells differentiate into mineralizing cells, and the bridging of the fractured ends results in the bone union. The mineralized callus then undergoes remodeling to restore the original shape and structure of the healed bone. Fracture healing has been studied in mice using various injury models. Still, the best way to recapitulate this entire biological process is to break through the cross-section of a long bone that encompasses both cortices. This protocol describes how a stabilized, transverse femur fracture can be safely performed to assess healing in adult mice. A surgical protocol including detailed harvesting and imaging techniques to characterize the different stages of fracture healing is also provided.

This protocol describes a method to perform fractures on adult mice and monitor the healing process.

Fratura transversa do fêmur do rato com pino estabilizador

Fracture repair is an essential function of the skeleton that cannot be reliably modeled in vitro. A mouse injury model is an efficient approach to test ...

A Modified Technique for Transverse Aortic Constriction in Mice

Transverse aortic constriction (TAC) is a frequently used surgery in research regarding heart failure and cardiac hypertrophy based on the formation of pressure overload in mouse models. The main challenge of this procedure is to clearly visualize the transverse aortic arch and precisely band the target vessel. Classical approaches perform a partial thoracotomy to expose the transverse aortic arch. However, it is an open-chest model that causes a rather large surgical trauma and requires a ventilator during the surgery. To prevent unnecessary trauma and simplify the operating procedure, the aortic arch is approached via the proximal proportion of the sternum, reaching and binding the target vessel using a small self-made retractor that contains a snare. This procedure can be conducted without entering the pleura cavity and does not need a ventilator or microsurgical operation, which leaves the mice with physiological breathing patterns, simplifies the procedure, and significantly reduces operation time. Due to the less invasive approach and less operation time, mice can undergo fewer stress reactions and recover rapidly.

The present protocol describes a modified and simplified technique with a minimally invasive transverse aortic constriction (TAC) procedure using a self-made retractor. This procedure can be conducted without a ventilator or microscope and introduces pressure overload, eventually leading to cardiac hypertrophy or heart failure.

Técnica Modificada para Constrição Aórtica Transversa em Camundongos

Transverse aortic constriction (TAC) is a frequently used surgery in research regarding heart failure and cardiac hypertrophy based on the formation of ...

A Calcium Phosphate-Induced Mouse Abdominal Aortic Aneurysm Model

An abdominal aortic aneurysm (AAA) is a life-threatening cardiovascular disease that occurs worldwide and is characterized by irreversible dilation of the abdominal aorta. Currently, several chemically induced murine AAA models are used, each simulating a different aspect of the pathogenesis of AAA. The calcium phosphate-induced AAA model is a rapid and cost-effective model compared to the angiotensin II- and elastase-induced AAA models. The application of CaPO4 crystals to the mouse aorta results in elastic fiber degradation, loss of smooth muscle cells, inflammation, and calcium deposition associated with aortic dilation. This article introduces a standard protocol for the CaPO4-induced AAA model. The protocol includes material preparation, the surgical application of the CaPO4&#160;to the adventitia of the infrarenal abdominal aorta, the harvesting of aortas to visualize aortic aneurysms, and histological analyses in mice.

This protocol describes a calcium phosphate-induced abdominal aortic aneurysm (AAA) mouse model to study the pathological features and molecular mechanisms of AAAs.

Um Modelo de Aneurisma da Aorta Abdominal de Camundongo Induzido por Fosfato de Cálcio

An abdominal aortic aneurysm (AAA) is a life-threatening cardiovascular disease that occurs worldwide and is characterized by irreversible dilation of the ...