Name: a murine model of arterial restenosis: technical aspects of femoral wire injury
Uploaded: 2015-03-10T15:00:00.000+00:00
Duration: 6 min 42 s
Description: Watch this Scientific Journal Video about A Murine Model of Arterial Restenosis: Technical Aspects of Femoral Wire Injury at JoVE.com

This work was supported by a Wisconsin Partnership Program New Investigator Award (ID 2832), a National Heart, Lung, Blood Institute R01 Grant (HL-068673) and a T32 training Grant (HL-110853). We thank Dr. Melina Kibbe&#8217;s group at Northwestern University for providing helpful information.

Declaração de Ética: NOTA: Todos os procedimentos estão em conformidade com o Manual sobre Cuidados e Uso de Animais de Laboratório (National Academies Press, 8ª edição, 2011), e os protocolos aprovados pelo Comitê de Uso e Cuidado Animal Institucional da Universidade de Wisconsin. Todas as cirurgias foram realizadas sob anestesia isoflurano (através de inalação, taxa de fluxo de 2 ml / min), e foram feitos todos os esforços para minimizar o sofrimento. Os animais foram sacrificados numa câmara gradualmente cheio com CO 2. 1. A indução da anestesia geral e incisão na pele <ol><li> Use macho C57BL / 6 com idade de 12-16 semanas, com peso de 23-28 g. Anestesiar o animal por isoflurano através de um aparelho anestésico-circuito fechado. Confirme anesthetization adequada por beliscar seu dedo do pé. Aplique um pouco de pomada lágrimas artificiais nos olhos para evitar o ressecamento. </li><li> Posicione o mouse em decúbito dorsal com o membro posterior esquerdo ligeiramente sequestrado e do joelho sl conjuntaightly flexionados. Raspar o cabelo em torno da região da coxa esquerda e desinfectar a pele com clorexidina, aplicado três vezes usando algodão estéril aplicadores derrubado. Use um esterilizador talão quente para manter os instrumentos esterilizados durante a cirurgia. </li><li> Coloque uma cortina de gaze esterilizada sobre a perna esquerda. Faça, uma incisão na pele longitudinal 1,5 centímetros em linha reta na coxa medial esquerdo da articulação do joelho até a virilha (Figura 1A). </li></ol> 2. Exposição da artéria femoral e Ramos NOTA: A Figura 1B mostra a anatomia vascular grave na coxa superior. A artéria femoral superficial (SFA) pode ser observado através de um microscópio de dissecação, quando a incisão na pele é adequadamente feita ao longo da linha média da coxa medial. <ol><li> Abra-se a fáscia fina que recobre a SFA com uma pinça fina e, em seguida, enrole o SFA com 9-0 nylon sutura. NOTA: Neste processo, o nervo femoral é facilmente colocar comoide, mas a veia femoral adjacente requer muita atenção, pois sua parede é tão fina que facilmente perfura e sangra. A técnica chave é se concentrar na própria artéria; enquanto a dissecção é realizada perto da artéria, veia qualquer dano acidental é evitável. </li><li> Uma vez que a SFA é enrolado, segure o fio de sutura com uma pinça mosquito e puxe-o levemente para baixo. Essa manobra torna ainda mais dissecção mais fácil, mas para evitar danificar a artéria não puxar com muita força. </li><li> Continue dissecção da artéria proximal para expor e laço da artéria femoral comum (CFA) com 9-0 nylon sutura. NOTA: Normalmente, o looping CFA em um ponto distal à artéria epigástrica (Figura 1B) é suficiente para a inserção do fio. Segure o laço de sutura CFA com uma pinça mosquito da mesma forma. </li><li> Puxe o laço de sutura CFA para cima e para o laço de sutura SFA para baixo para descobrir a bifurcação da SFA e artéria femoral profunda (DFA). O DFA, o principal ramo do CFA,vai para o músculo da coxa (Figura 1B). NOTA: O fio será inserido a partir da DFA mais tarde. </li><li> Passe o DFA com cuidado extra porque a veia adjacente está embaraçada por baixo da artéria. Em caso de hemorragias a partir da veia, um de 30 a 60 segundos de compressão simples com gaze é geralmente suficiente para produzir a hemostasia. </li><li> Uma vez que o DFA é intercalado com 9-0 nylon sutura, certifique-se todos os três laços de sutura estão em suas posições corretas (Figura 2A). </li><li> Prossiga com a dissecção da DFA distal ao ligar a artéria em um ponto, na medida do possível a partir da bifurcação. Cortar sutura extra, mas deixe um determinado comprimento de modo que as duas extremidades pode ser realizada com uma pinça de mosquito, que estabilizam o DFA tornando mais fácil a inserção do fio. </li></ol> 3. Criação de Lesão Fio <ol><li> Insira fio via DFA <ol><li> Coe as suturas CFA, SFA, e DFA para estabelecer uma pinça hemostática temporária. </li><li> Faça um pequeno furo no DFUm com microtesoura. Inserir as pontas das micropinças curvas para dilatar o orifício suficientemente grande para um fio com um diâmetro de 0,015 polegadas (Figura 2B demonstra a diferença de tamanho entre a artéria e o fio). <ol><li> Antes de fazer um buraco na DFA, afrouxar o laço de sutura CFA e gentilmente apertar / apertar o DFA várias vezes com micropinças maçantes de ponta. Esta manobra aumenta o diâmetro arterial notavelmente para facilitar a inserção do fio. Humedecer o fio cuidadosamente com PBS também é importante para uma inserção suave. </li><li> Ao fazer um buraco na DFA, não colhe a parede arterial, mas fazer uma pequena alfinetada em forma de V e beliscar a aba com micropinças de ponta aguçada para a manipulação. </li></ol></li><li> Insira o fio através do furo adequadamente-dilatada no DFA. Gentilmente avançar o fio em direção à artéria ilíaca, enquanto soltando o laço de sutura em torno do CFA. 5-8 mm é suficiente para cobrir todo o comprimento CFA na maioria dos casos (Figura 2C </strong&gt;). Não insira o fio com força como uma perfuração arterial geralmente resulta em perda do mouse. Encurtar o arame para cerca de 15 mm de comprimento ajuda com a manipulação. </li></ol></li><li> Deixar fio dentro de um Min <ol><li> Depois de inserir o fio com o comprimento óptimo, deixá-lo durante 1 min para esticar a parede arterial. Umedeça o fio e artérias com PBS para evitar a secagem. Outra sutura de nylon 9-0 pode ser colocada em torno da DFA proximal ao ponto de inserção de fio durante este tempo de espera. Esta sutura será utilizado mais tarde para fechar o buraco no DFA após a remoção do fio. </li></ol></li><li> Recuperar fio e retomar o fluxo de sangue <ol><li> Segure o fio com uma pinça de punção embarcação, e puxe-o para trás suavemente; o fio deve ser recuperada sem problemas. Para ultrapassar qualquer resistência, geralmente causado pela adesão do arame para a parede arterial, puxando para trás o fio com uma rotação suave é útil. Mais uma vez, uma manobra forçada poderia resultar em perda de animal. </li><li> AD epois de recuperar o fio, ligar o DFA no ponto de bifurcação com sutura de nylon 9-0. Não ligar muito próximo à bifurcação, o que comprometeria a saída de sangue para o SFA. </li><li> Solte os laços de sutura SFA e CFA para retomar o fluxo de sangue normal. Note-se que o CFA é bem expandido pela inserção de fio (Figura 2D). </li></ol></li></ol> 4. Pele Encerramento e Cuidados Pós-Operatórios <ol><li> Controle sangra. Exsudação Minor pode ser interrompido por compressão simples; algumas grandes hemorragias arteriais vai precisar de pontos com uma sutura 11-0 nylon. Estas artérias suturadas, acabará por resultar em oclusão arterial. </li><li> Feche a pele ferida com grampeadores da pele (geralmente 3-4 grampos). Mantenha a área ferida esterilizado através da aplicação de solução cirúrgica clorexidina. </li><li> Aplicar tratamentos pós-cirúrgicos. Controlar a dor do mouse com injeções subcutâneas de buprenorfina. Mantenha o mouse sobre a almofada de aquecimento até que recuperou sufficiente de consciência para manter decúbito esternal. Casa-lo em uma gaiola limpa sem a companhia de outros animais até que esteja totalmente recuperado. </li></ol>

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	<li>Dzau, V. J., Braun-Dullaeus, R. C., Sedding, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+proliferation+and+atherosclerosis:+new+perspectives+and+therapeutic+strategies.">Vascular proliferation and atherosclerosis: new perspectives and therapeutic strategies.</a> Nat Med. 8 (11), 1249-1256 (2002).</li><li>Hui, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intimal+hyperplasia+in+murine+models.">Intimal hyperplasia in murine models.</a> Curr Drug Targets. 9 (3), 251-260 (2008).</li><li>Ferns, G. A., Avades, T. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanisms+of+coronary+restenosis:+insights+from+experimental+models.">The mechanisms of coronary restenosis: insights from experimental models.</a> Int J Exp Pathol. 81 (2), 63-88 (2000).</li><li>Sata, 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=A+mouse+model+of+vascular+injury+that+induces+rapid+onset+of+medial+cell+apoptosis+followed+by+reproducible+neointimal+hyperplasia.">A mouse model of vascular injury that induces rapid onset of medial cell apoptosis followed by reproducible neointimal hyperplasia.</a> J Mol Cell Cardiol. 32 (11), 2097-2104 (2000).</li><li>Xu, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+arteriosclerosis:+from+arterial+injuries+to+vascular+grafts.">Mouse models of arteriosclerosis: from arterial injuries to vascular grafts.</a> Am J Pathol. 165 (1), 1-10 (2004).</li><li>Lv, L., Meng, Q., Ye, M., Wang, P., Xue, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STAT4+deficiency+protects+against+neointima+formation+following+arterial+injury+in+mice.">STAT4 deficiency protects against neointima formation following arterial injury in mice.</a> J Mol Cell Cardiol. 74C, 284-294 (2014).</li><li>Zhu, B., Kuhel, D. G., Witte, D. P., Hui, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apolipoprotein+E+inhibits+neointimal+hyperplasia+after+arterial+injury+in+mice.">Apolipoprotein E inhibits neointimal hyperplasia after arterial injury in mice.</a> Am J Pathol. 157 (6), 1839-1848 (2000).</li><li>Fu, Y., Duru, E. A., Davies, M. G. <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+metabolic+syndrome+on+the+response+to+arterial+injury.">Effect of metabolic syndrome on the response to arterial injury.</a> J Surg Res. , (2014).</li><li>Kuhel, D. G., Zhu, B., Witte, D. P., Hui, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinction+in+genetic+determinants+for+injury-induced+neointimal+hyperplasia+and+diet-induced+atherosclerosis+in+inbred+mice.">Distinction in genetic determinants for injury-induced neointimal hyperplasia and diet-induced atherosclerosis in inbred mice.</a> Arterioscler Thromb Vasc Biol. 22 (6), 955-960 (2002).</li><li>Cooley, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+strain+differential+neointimal+response+in+vein+grafts+and+wire-injured+arteries.">Mouse strain differential neointimal response in vein grafts and wire-injured arteries.</a> Circ J. 71 (10), 1649-1652 (2007).</li><li>Ishigami, N., 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=Deficiency+of+CuZn+superoxide+dismutase+promotes+inflammation+and+alters+medial+structure+following+vascular+injury.">Deficiency of CuZn superoxide dismutase promotes inflammation and alters medial structure following vascular injury.</a> J Atheroscler Thromb. 18 (11), 1009-1017 (2011).</li></ol>

The authors declare no competing financial interests.

O procedimento de lesão fio é aplicável a todas as linhagens de camundongos, desde que as suas estruturas anatômicas são semelhantes. 4 Neste trabalho, utilizou-se do sexo masculino camundongos C57BL / 6 com uma idade de 12-16 semanas. Como aprendemos a partir de nossos estudos preliminares, as artérias femorais de ratos mais jovens de 10 semanas são muitas vezes tão pequeno que a inserção do fio é bastante desafiador. Por outro lado, a inserção do fio nos ratos mais velhos que 16 semanas, é tec...

In the era of expanded application of endovascular treatments for various cardiovascular diseases, restenosis after angioplasty is one of the major problems for patients undergoing such treatments. Damage to the vascular endothelium at the time of angioplasty, in concert with the atherosclerotic background, induces excessive smooth muscle cell proliferation in the medial layer, resulting in neointimal hyperplasia.1,2 A viable animal model that recapitulates post-angioplasty neointimal hyperplasia is, thus, important not only for the investigation of disease mechanisms but also for the development of effective therapeutics to treat this pathology.
Mice represent an excellent model animal to recapitulate neointimal hyperplasia for the following reasons: the genetic backgrounds of experimental mice are well established; a wide variety of genetically-modified strains are available;2 obtaining littermates of the same background is easy; and the cost of the animals is relatively low. Arterial ligation model and wire injury model are the two most common mouse models of mechanically-induced neointimal hyperplasia. The arterial ligation model is easy to create, but physiologically dissimilar to the actual angioplasty procedure. The wire injury model closely mimics actual angioplasty procedures but is technically difficult due to the small size of mouse arteries.2,3 Sata et al. first described a wire injury method for mouse femoral arteries based on the anatomical structure of the vasculature and the use of proper-sized flexible wire. Utilizing this technique, they succeeded in reproducibly inducing neointimal hyperplasia in various strains of mice.4
Although femoral wire injury is a well-established model, some of the technical aspects of the technique are highly challenging compared to other models such as ligation.5 The purpose of this paper is to describe our mouse wire injury model procedures in detail, which is a modified version of Sata&#8217;s original method. We have made two main modifications: 1) Looping only the arteries, and 2) No lidocaine use.

<table><tbody><tr><td>Fixed Core Wire Guide</td><td>Cook</td><td>G02426</td><td>Diameter 0.015 inch, Straight</td></tr><tr><td>Dilation Forceps</td><td>Roboz</td><td>RS-4927</td><td>Curved, blunt tips</td></tr><tr><td>Dumont Tweesers #5</td><td>World Precision Instruments</td><td>14095</td><td>Straight, sharp tips</td></tr><tr><td>Dumont Vessel Cannulation Forceps</td><td>World Precision Instruments</td><td>503373</td><td></td></tr><tr><td>McPherson-Vannas Scissors</td><td>World Precision Instruments</td><td>501234</td><td></td></tr><tr><td>Mosquito Forceps</td><td>World Precision Instruments</td><td>501291</td><td></td></tr><tr><td>Ethilon Nylon Suture 9-0</td><td>Ethicon</td><td>7717G</td><td>9-0, Black Nylon Monofilament</td></tr><tr><td>Micro AROSuture, Sterile 11-0, 70 micrometers, MET Point</td><td>AROSurgical</td><td>VT4A00N07</td><td>11-0, Black Nylon Monofilament</td></tr><tr><td>3M Precise Multi-Shot DS Disposable Skin Stapler</td><td>3M</td><td>DS-25</td><td></td></tr></tbody></table>

a murine model of arterial restenosis: technical aspects of femoral wire injury

A doença cardiovascular causadas por aterosclerose é a principal causa de morte no mundo desenvolvido. O estreitamento da luz do vaso, devido ao desenvolvimento da placa aterosclerótica ou a ruptura de placas estabelecidos, interrompe o fluxo normal do sangue levando a várias morbidades, tais como infarto do miocárdio e acidente vascular cerebral. Na clínica procedimentos endovasculares, como a angioplastia são comumente realizados para reabrir o lúmen. No entanto, estes tratamentos inevitavelmente danificar a parede do vaso, bem como o endotélio vascular, desencadeando uma resposta de cicatrização excessiva e o desenvolvimento de uma placa que se estende da neointima no lúmen fazendo com que a restenose do vaso (re-estreitamento). A reestenose permanece uma das principais causas do fracasso dos tratamentos endovasculares para aterosclerose. Assim, os modelos animais pré-clínicos de reestenose são de vital importância para a investigação dos mecanismos fisiopatológicos, bem como translacional abordagens para intervenções vasculares. Entre vários experime murinomodelos ntal, lesão fio artéria femoral é amplamente aceito como o mais adequado para estudos de reestenose pós-angioplastia, pois se assemelha ao procedimento de angioplastia que prejudica a ambos endotélio e da parede do vaso. No entanto, muitos pesquisadores têm dificuldade em utilizar este modelo devido ao seu alto grau de dificuldade técnica. Isto é primeiramente porque um fio de metal tem de ser inserido dentro da artéria femoral, o que é cerca de três vezes mais fino do que o fio, para gerar suficiente para induzir lesão neoíntima proeminente. Aqui, descrevemos os detalhes cirúrgicos essenciais para efetivamente superar as maiores dificuldades técnicas deste modelo. Seguindo os procedimentos apresentados, realizando a lesão de arame artéria femoral de rato torna-se mais fácil. Uma vez familiarizado, todo o processo pode ser concluído no prazo de 20 min.

Quatro semanas após a cirurgia, dissecção da artéria femoral feridos fios e fazer espécime Bloco A parafina para analisar a formação neointima. Induzir anestesia geral, tal como descrito acima. Abra o peito imediatamente e insira uma de calibre 20 borboleta agulha para o ventrículo esquerdo. Ligue a linha para um frasco de infusão. Realizam a fixação de perfusão através de infusão de gotas de PBS, seguido por paraformaldeído a 4% (geralmente 10-20 mL, cada um). O frasco de infusão é colocado ce...

Watch this Scientific Journal Video about A Murine Model of Arterial Restenosis: Technical Aspects of Femoral Wire Injury at JoVE.com

A Murine Model of Arterial Restenosis: Technical Aspects of Femoral Wire Injury

O mouse modelo de lesão fio artéria femoral de reestenose é tecnicamente desafiador. Neste protocolo, mostrar os principais detalhes técnicos essencial para a realização com êxito lesão fio para induzir neoï¿½tima consistente para estudos de reestenose.

Um modelo murino de Arterial Restenosis: Aspectos Técnicos do Femoral Injury Fio

Cardiovascular disease caused by atherosclerosis is the leading cause of death in the developed world. Narrowing of the vessel lumen, due to ...

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Research

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Medicine

20.1K visualizações.  University of Wisconsin School of Medicine and Public Health.  O mouse modelo de lesão fio artéria femoral de reestenose é tecnicamente desafiador. Neste protocolo, mostrar os principais detalhes técnicos essencial para a realização com êxito lesão fio para induzir neoï¿½tima consistente para estudos de reestenose. 

Vídeo: A Murine Model of Arterial Restenosis: Technical Aspects of Femoral Wire Injury

Murine Model of Hindlimb Ischemia 

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide. Medical therapies for symptomatic relief are limited. Surgical or endovascular interventions are useful for some individuals, but long-term results are often disappointing. As a result, there is a need for developing new therapies to treat PAD. The murine hindlimb ischemia preparation is a model of PAD, and is useful for testing new therapies. When compared to other models of tissue ischemia such as coronary or cerebral artery ligation, femoral artery ligation provides for a simpler model of ischemic tissue. Other advantages of this model are the ease of access to the femoral artery and low mortality rate. 
In this video, we demonstrate the methodology for the murine model of unilateral hindimb ischemia. The specific materials and procedures for creating and evaluating the model will be described, including the assessment of limb perfusion by laser Doppler imaging. This protocol can also be utilized for the transplantation and non-invasive tracking of cells, which is demonstrated by Huang et al.1.

Murine Model of Hindlimb Ischemia

The surgical procedure for induction of unilateral hindlimb ischemia is demonstrated, with confirmation of ischemia by laser Doppler perfusion imaging.

Modelo murino de isquemia dos membros posteriores

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide.  Medical therapies for ...

Biologia

Murine Spinotrapezius Model to Assess the Impact of Arteriolar Ligation on Microvascular Function and Remodeling

The murine spinotrapezius is a thin, superficial skeletal support muscle that extends from T3 to L4, and is easily accessible via dorsal skin incision. Its unique anatomy makes the spinotrapezius useful for investigation of ischemic injury and subsequent microvascular remodeling. Here, we demonstrate an arteriolar ligation model in the murine spinotrapezius muscle that was developed by our research team and previously published1-3. For certain vulnerable mouse strains, such as the Balb/c mouse, this ligation surgery reliably creates skeletal muscle ischemia and serves as a platform for investigating therapies that stimulate revascularization. Methods of assessment are also demonstrated, including the use of intravital and confocal microscopy. The spinotrapezius is well suited to such imaging studies due to its accessibility (superficial dorsal anatomy) and relative thinness (60-200 &mu;m). The spinotrapezius muscle can be mounted en face, facilitating imaging of whole-muscle microvascular networks without histological sectioning. We describe the use of intravital microscopy to acquire metrics following a functional vasodilation procedure; specifically, the increase in arterilar diameter as a result of muscle contraction. We also demonstrate the procedures for harvesting and fixing the tissues, a necessary precursor to immunostaining studies and the use of confocal microscopy.

We demonstrate a novel arterial ligation model in murine spinotrapezius muscle, including a step-by-step procedure and description of required instrumentation. We describe the surgery and relevant outcome measurements relating to vascular network remodeling and functional vasodilation using intravital and confocal microscopy.

Modelo Spinotrapezius murino para avaliar o impacto da ligadura arteriolar em função microvascular e Remodelação

The murine spinotrapezius is a thin, superficial skeletal support muscle that extends from T3 to L4, and is easily accessible via dorsal skin incision. ...

Medicina

A Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis

Despite the considerable progress made in the stent development in the last decades, cardiovascular diseases remain the main cause of death in western countries. Beside the benefits offered by the development of different drug-eluting stents, the coronary revascularization bears also the life-threatening risks of in-stent thrombosis and restenosis. Research on new therapeutic strategies is impaired by the lack of appropriate methods to study stent implantation and restenosis processes. Here, we describe a rapid and accessible procedure of stent implantation in mouse carotid artery, which offers the possibility to study in a convenient way the molecular mechanisms of vessel remodeling and the effects of different drug coatings.

A model of stent implantation in mouse carotid artery is described. Compared to other similar methods, this procedure is very rapid, simple and accessible, offering the possibility to study in a convenient way the vascular wall reaction to different drug-eluting stents and the molecular mechanisms of restenosis.

Um modelo murino de implante de stent na artéria carótida para o Estudo da reestenose

Despite  the considerable progress made in the stent development in the last decades,  cardiovascular diseases remain the main cause of death in western ...

Generation and 3-Dimensional Quantitation of Arterial Lesions in Mice Using Optical Projection Tomography

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating important information on the pathogenesis of lesion formation and the action of novel therapies. Use of atherosclerosis-prone mice, surgical methods of lesion induction, and dietary modification has dramatically improved understanding of the mechanisms that contribute to disease development and the potential of new treatments.
Classically, analysis of lesions is performed ex vivo using 2-dimensional histological techniques. This article describes application of optical projection tomography (OPT) to 3-dimensional quantitation of arterial lesions. As this technique is non-destructive, it can be used as an adjunct to standard histological and immunohistochemical analyses.
Neointimal lesions were induced by wire-insertion or ligation of the mouse femoral artery whilst atherosclerotic lesions were generated by administration of an atherogenic diet to apoE-deficient mice.
Lesions were examined using OPT imaging of autofluorescent emission followed by complementary histological and immunohistochemical analysis. OPT clearly distinguished lesions from the underlying vascular wall. Lesion size was calculated in 2-dimensional sections using planimetry, enabling calculation of lesion volume and maximal cross-sectional area. Data generated using OPT were consistent with measurements obtained using histology, confirming the accuracy of the technique and its potential as a complement (rather than alternative) to traditional methods of analysis.
This work demonstrates the potential of OPT for imaging atherosclerotic and neointimal lesions. It provides a rapid, much needed ex vivo technique for the routine 3-dimensional quantification of vascular remodelling.

Ex vivo analysis of arterial lesions from animal models of cardiovascular disease classically relies on histological and immunohistochemical techniques. These provide 2-dimensional measurements in 3-dimensional lesions. This manuscript describes the generation of arterial lesions for quantitative analysis in 3-dimensions using optical projection tomography.

Geração e quantificação 3-Dimensional de Arterial lesões em ratos por meio de tomografia óptica de Projeção

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating ...

Murine Model of Femoral Artery Wire Injury with Implantation of a Perivascular Drug Delivery Patch

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. While these therapies lead to the rapid restoration of blood flow, these technologies remain limited by restenosis in the case of bare metal stents and angioplasty, or reduced healing and possibly enhanced risk of thrombosis in the case of drug eluting stents. A key pathophysiological mechanism in the formation of restenosis is intimal hyperplasia caused by the activation of vascular smooth muscle cells and inflammation due to arterial stretch and injury. Surgeries that induce arterial injury in genetically modified mice are useful for the mechanistic study of the vascular response to injury but are often technically challenging to perform in mouse models due to the their small size and lack of appropriate sized devices. We describe two approaches for a surgical technique that induces endothelial denudation and arterial stretch in the femoral artery of mice to produce robust neointimal hyperplasia. The first approach creates an arteriotomy in the muscular branch of the femoral artery to obtain vascular access. Following wire injury this arterial branch is ligated to close the arteriotomy. A second approach creates an arteriotomy in the main femoral artery that is later closed through localized cautery. This method allows for vascular access through a larger vessel and, consequently, provides a less technically demanding procedure that can be used in smaller mice. Following either method of arterial injury, a degradable drug delivery patch can be placed over or around the injured artery to deliver therapeutic agents.

We describe a surgical technique that produces wire injury in the femoral artery of mice to induce neointimal hyperplasia to serve as a model testing system for the perivascular delivery of therapeutic compounds for the inhibition of restenosis.

Modelo murino de artéria femoral Injury fio com a implantação de uma droga patch Entrega perivascular

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

Indução de Aterosclerose Acelerada em Camundongos: O Modelo de "Lesão por Fio"

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Multiphoton Intravital Imaging for Monitoring Leukocyte Recruitment during Arteriogenesis in a Murine Hindlimb Model

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for analyzing collateral arteries and leukocytes in arteriogenesis is ex vivo (immuno-) histological methodology. However, this technique does not allow the measurement of dynamic processes such as blood flow, shear stress, cell-cell interactions, and particle velocity. This paper presents a protocol to monitor in vivo processes in growing collateral arteries during arteriogenesis utilizing intravital imaging. The method described here is a reliable tool for dynamics measurement and offers a high-contrast analysis with minimal photo-cytotoxicity, provided by multiphoton excitation microscopy. Prior to analyzing growing collateral arteries, arteriogenesis was induced in the adductor muscle of mice by unilateral ligation of the femoral artery.
After the ligation, the preexisting collateral arteries started to grow due to increased shear stress. Twenty-four hours after surgery, the skin and subcutaneous fat above the collateral arteries were removed, constructing a pocket for further analyses. To visualize blood flow and immune cells during in vivo imaging, CD41-fluorescein isothiocyanate (FITC) (platelets) and CD45-phycoerythrin (PE) (leukocytes) antibodies were injected intravenously (i.v.) via a catheter placed in the tail vein of a mouse. This article introduces intravital multiphoton imaging as an alternative or in vivo complementation to the commonly used static ex vivo (immuno-) histological analyses to study processes relevant for arteriogenesis. In summary, this paper describes a novel and dynamic in vivo method to investigate immune cell trafficking, blood flow, and shear stress in a hindlimb model of arteriogenesis, which enhances evaluation possibilities notably.

The recruitment of leukocytes and platelets constitutes an essential component necessary for the effective growth of collateral arteries during arteriogenesis. Multiphoton microscopy is an efficient tool for tracking cell dynamics with high spatio-temporal resolution in vivo and less photo-toxicity to study leukocyte recruitment and extravasation during arteriogenesis.

Imagem Intravital Multifóton para Monitoramento do Recrutamento de Leucócitos durante a Arteriogênese em Modelo de Membro Posterior Murino

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for ...

High-Resolution Three-Dimensional Imaging of the Footpad Vasculature in a Murine Hindlimb Gangrene Model

Peripheral arterial disease (PAD) is a significant cause of morbidity resulting from chronic exposure to atherosclerotic risk factors. Patients suffering from its most severe form, chronic limb-threatening ischemia (CLTI), face substantial impairments to daily living, including chronic pain, limited walking distance without pain, and nonhealing wounds. Preclinical models have been developed in various animals to study PAD, but mouse hindlimb ischemia remains the most widely used. There can be significant variation in response to ischemic insult in these models depending on the mouse strain used and the site, number, and means of arterial disruption. This protocol describes a unique method combining femoral artery and vein electrocoagulation with the administration of a nitric oxide synthase (NOS) inhibitor to reliably induce footpad gangrene in Friend Virus B (FVB) mice that resembles the tissue loss of CLTI. While traditional means of assessing reperfusion such as laser Doppler perfusion imaging (LDPI) are still recommended, intracardiac perfusion of the lipophilic dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) is used to label the vasculature. Subsequent whole-mount confocal laser scanning microscopy allows for high-resolution, three-dimensional (3D) reconstruction of footpad vascular networks that complements traditional means of assessing reperfusion in hindlimb ischemia models.

The present protocol describes a unique, clinically relevant model of peripheral arterial disease that combines femoral artery and vein electrocoagulation with the administration of a nitric oxide synthase inhibitor to induce hindlimb gangrene in FVB mice. Intracardiac DiI perfusion is then used for high-resolution, three-dimensional imaging of the footpad vasculature.

Imagem tridimensional de alta resolução da Vasculatura do Footpad em um Modelo de Gangrena Hindlimb Murine

Peripheral arterial disease (PAD) is a significant cause of morbidity resulting from chronic exposure to atherosclerotic risk factors. Patients suffering ...

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell (VSMC) proliferation. Common murine models study small vessels such as the carotid and femoral arteries. Herein we describe an in vivo system in which both VSMC proliferation and endothelial barrier function can be simultaneously assessed in a large vessel. We studied the infrarenal aortic response to injury in C57BL/6 mice. The aorta was injured from the left renal vein to the aortic bifurcation by 30 transmural crushes of 5-seconds duration with a cotton-tipped applicator. Morphological changes were assessed with conventional histology. Aorta wall thickness was measured from the luminal surface to the adventitia. EdU integration and counter staining with DAPI and alpha-actin was used to demonstrate VSMC proliferation. Activation of ERK1/2, a known moderator of intimal hyperplasia formation, was determined by Western Blot analysis. The effect of inflammation was determined by immunohistochemistry for B-cells, T-cells, and macrophages. En face sections of endothelium were visualized with scanning electron microscopy (SEM). Endothelial barrier function was determined with Evans Blue staining. Transmural injury resulted in aortic wall thickening. This injury induced VSMC proliferation, most prominently at 3 days after injury, and early activation of ERK1/2 and decreased p27kip1 expression. Injury did not result in increased B-cells, T-cells, or macrophages infiltration in the vessel wall. Injury caused partial endothelial cell denudation and loss of cell-cell contact. Injury resulted in a significant loss of endothelial barrier function, which returned to baseline after seven days. The murine transmural blunt aortic injury model provides an efficient system to simultaneously study both VSMC proliferation and endothelial barrier function in a large vessel.

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Restenosis following cardiovascular procedures (bypass surgery, angioplasty, or stenting) is a significant problem reducing the durability of these procedures. An ideal therapy would inhibit smooth muscle cell (VSMC) proliferation while promoting regeneration of the endothelium. We describe a model for simultaneous assessment of VSMC proliferation and endothelial function in vivo.

Lesão por esmagamento aórtico murino: um eficiente<em&gt; In Vivo</em&gt; Modelo de proliferação de células musculares lisas e função endotelial

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell ...

Establishing a Diaphyseal Femur Fracture Model in Mice

Bones have a significant regenerative capacity. However, fracture healing is a complex process, and depending on the severity of the lesions and the age and overall health status of the patient, failures can occur, leading to delayed union or nonunion. Due to the increasing number of fractures resulting from high-energy trauma and aging, the development of innovative therapeutic strategies to improve bone repair based on the combination of skeletal/mesenchymal stem/stromal cells and biomimetic biomaterials is urgently needed. To this end, the use of reliable animal models is fundamental to better understanding the key cellular and molecular mechanisms that determine the healing outcomes. Of all the models, the mouse is the preferred research model because it offers a wide variety of transgenic strains and reagents for experimental analysis. However, the establishment of fractures in mice may be technically challenging due to their small size. Therefore, this article aims to demonstrate the procedures for the surgical establishment of a diaphyseal femur fracture in mice, which is stabilized with an intramedullary wire and resembles the most common bone repair process, through cartilaginous callus formation.

This protocol describes a surgical procedure for the establishment of a diaphyseal fracture in the femur of mice, which is stabilized with an intramedullary wire, for fracture healing studies.

Estabelecimento de um Modelo de Fratura Diafisária do Fêmur em Camundongos

Bones have a significant regenerative capacity. However, fracture healing is a complex process, and depending on the severity of the lesions and the age ...