Os autores agradecem Brian Johnson da Histologia e imagem Núcleo da Universidade de Washington para obter ajuda com a coloração tricromo e análise. Este trabalho foi financiado pelo NIH concede HL098067 e HL089455.

Os Comitês Institucionais de Animais Cuidado e Uso (IACUC) da Universidade de Washington e Cedars-Sinai Medical Center aprovaram o animal de trabalho necessário para estes estudos. 1. Preparação <ol><li> Esterilizar ambas as pinças de extremidade embotada e o autoclave através de depressor. </li><li> Usando uma câmara de segurança biológica, prepare um estoque de trabalho de BLM em PBS a partir do pó liofilizado. Sonicar a solução durante 10 min a 35 kHz a assegurar uma mistura uniforme. Nota: Um volume total de entre 30 e 45 uL é recomendado para evitar a variação de pipetagem na extremidade baixa, e asfixia com volumes maiores. </li><li> Prepare um espaço de trabalho limpo, que inclui cerca de 1 m 2 para o procedimento em si, bem como locais designados para gaiolas antes e após o procedimento. </li><li> Fixar a placa de base do procedimento para o banco imediatamente em frente do investigador por a disposição de 2 ou 3 tiras de fita laboratório através da base e Sublinharying banco. Veja a Figura 1 para mais especificações sobre a criação de um conselho. </li><li> Amarre um único comprimento de tamanho 4.0 fio de sutura entre os dois parafusos de posicionamento da placa de procedimento. </li><li> Gerar um espirómetro improvisada, removendo e rejeitando o êmbolo a partir de três seringas de 1 ml, e depositar 60 ul de PBS para o topo de cada barril, para formar uma vedação hermética. Fixar o cubo do cateter livremente para uma das seringas e colocá-lo para um lado do tabuleiro. </li><li> Aspirar 300 ul de ar para uma seringa de 1 mL e colocá-lo para um lado do tabuleiro. </li><li> Cortar uma peça adicional de fita de aproximadamente 6 polegadas de comprimento e lugar para um lado. Isso será usado para prender o animal para o conselho na etapa 2.4. </li><li> Configurar uma câmara de isoflurano. Fixe O 2, isoflurano, e de vácuo para as portas apropriadas, tanto a câmara de exposição e o vácuo de apuramento. Em alternativa, administrar um anestésico de isoflurano-compatível biológicagabinete de segurança. </li></ol> 2. intubação <ol><li> Anestesiar o rato com isoflurano na câmara até que ela perde a consciência e respiração retarda a uma taxa adequada. Uma exposição típico inclui 4% de isoflurano e 2% de O 2 durante 3 a 4 minutos, e o resultado ideal é de 2 a 2,5 min da sedação. Isto corresponde a uma taxa de respiração de uma respiração cada 2 segundos. </li><li> Enquanto espera para a sedação para, em conjunto, aspirado entre 30 e 45 l de BLM em uma pipeta e lugar para um lado. </li><li> Quando estiver pronto, suspender o mouse sedado por seus incisivos superiores do fio ligado aos parafusos de posicionamento da plataforma procedimento. Certifique-se de que o dorso do animal fica plana contra a superfície da plataforma. </li><li> Tendo o cuidado de não restringir a ventilação, colocar um pedaço de fita livremente entre a parte inferior (caudal) da porção interior da cavidade torácica, logo acima do diafragma. A colocação deve ser apertado o suficiente para manter o alinhamento adequado durante o procedure, mas não tão apertado que restringe a respiração. </li><li> Ligue o iluminador para entre 80% e 100% de intensidade e orientar o pescoço de ganso de modo que é de 1 a 2 cm da superfície da pele, próximo do plexo solar. Verificar periodicamente a ponta do pescoço de ganso de calor para garantir que ele não ferir o rato. </li><li> De pé por trás da plataforma, use as estéreis, pinças fim brusco para localizar a língua. Ser cuidadoso para evitar os incisivos inferiores, suavemente aderência e desenhar a lingueta para fora da cavidade oral. </li><li> Usando a mão restante, insira o depressor e usá-lo para achatar a língua contra o assoalho da cavidade oral. Solte a pinça, mas deixe o depressor no lugar para as próximas duas etapas. </li><li> Orientar a luz de modo que a traqueia é visível, orientando o pescoço de ganso proximalmente a partir do nível do plexo solar até atingir o nível dos brônquios mainstem. Nota: A traqueia pode ser facilmente distinguidos pela acção da respiração, WHICH faz com que a luz emitida a flutuar em intensidade. Quando correctamente posicionada, esta estrutura será visível no plano axial de um pino localizado centralmente de luz com luz ambiente mínima na própria cavidade oral. </li><li> Ângulo da seringa para que ele segue o caminho natural da traqueia, e abaixe a ponta do cateter 22-G, com a seringa anexo contendo a gota, em linha reta para o lúmen. A bolha PBS começará a subir e descer com cada respiração em cima colocação bem sucedida. Nota: Esta acção pode ser retardado por vários segundos, como resultado da sedação profunda. </li><li> Alimentar o cateter em um adicional de 5 mm. Retire a espátula. </li><li> Deslocar a seringa ao lado oposto, e agarrando o hub, remova cuidadosamente a seringa. </li><li> Depósito entre 30 e 45 ul de BLM no centro do interior do cubo de cateter, anexar a segunda seringa e dispensar 300 ul de ar para dentro do cubo. </li><li> Substitua o segundoSeringa com o primeiro contendo a bolha de PBS. A bolha vai continuar a subir e descer se o procedimento foi realizado com sucesso. </li></ol> Cuidado 3. pós-procedimento <ol><li> Retirar o cateter e fita, e colocar o animal em um lugar quente e seco até que ele recupera a consciência - geralmente dentro de um par de minutos. </li></ol>

<ol>
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The authors have nothing to disclose.

Nos casos em que aerossolização é impraticável devido à disponibilidade limitada de reagentes, de segurança, ou de custos, a administração traqueal directa é um método superior para a entrega de agentes exógenos aos pulmões 16 transtraqueal instilação tem sido amplamente utilizada para realizar isto.; No entanto, como acontece com qualquer intervenção cirúrgica, ele também carrega consigo o potencial de complicações causadas pelo procedimento em si, e não necessariamente o agente a ser in...

Apesar de algumas diferenças anatômicas e fisiológicas, 1 modelos murino continuam a ser de valor inestimável para a modelagem de biologia humana e patogênese da doença. 2 Do ponto de vista pecuária, os ratos são fáceis de manusear, tem um tempo de reprodução baixa, um tempo de vida acelerado, e são relativamente baratos para abrigar. Com o desenvolvimento de diversas estirpes genéticas e estratégias (por ex., Condicionais knock-outs, ratinhos repórter, abordagens de rastreio de linhagem, etc.), bem como a grande variedade de reagentes disponíveis (por exemplo., Anticorpos, proteínas recombinantes, inibidores, etc.), os ratos se tornaram um organismo essencial modelo de vertebrados para descobrir processos de homeostase e de doenças humanas 3. Os ratos têm sido especialmente valiosa para estudar as condições pulmonares, incluindo lesão pulmonar aguda (LPA) e fibrose pulmonar. 4 LPA em seres humanos pode ser causado por trauma, lesão, ou septicemia e é caracterizada por células epiteliais evazamento endotelial (isto é., edema), inflamação e fibrose nascente. Em muitos pacientes, ALI progride para sua forma grave, síndrome do desconforto respiratório agudo (SDRA), que muitas vezes resulta em fibrose e morte por insuficiência respiratória. 5,6 fibrose pulmonar é uma patologia progressiva, fatal caracterizada pela deposição excessiva de matriz extracelular , digite mais notavelmente I colágeno, levando a função pulmonar prejudicada. 7,8 a administração de bleomicina (BLM) é o modelo mais amplamente utilizado e melhor caracterizado para induzir ALI e fibrose em animais experimentais. 9 Apesar de fibrose pulmonar BLM-induzida em roedores faz não recapitular plenamente os fenótipos fibróticas humanos, 10 estudos com ratos com este modelo levaram à descoberta de muitos fatores importantes que influenciam o aparecimento e progressão da doença 11. Enquanto o mecanismo exacto (s) por detrás fibrogénese induzida por BLM são desconhecidas, a lesão iniciarPensa-se que surgir a partir de rupturas dos filamentos de DNA dependente de contacto nas células epiteliais que revestem as vias aéreas condutoras e alvéolos e, em particular, tipo 1 pneumócitos. 12 A necessidade de contacto directo entre BLM eo epitélio pulmonar destaca a importância de uma rota de entrega robusta , e estas preocupações são também pertinentes para uma vasta gama de tratamentos direccionado para as vias respiratórias distais, incluindo proteínas recombinantes, anticorpos, siRNA, vírus, bactérias, partículas, e mais. Aspiração orofaríngea (OPA) tem sido amplamente utilizado para este fim 13, mas um grande inconveniente de uma OPA é que alguma porção do agente fornecido pode ser engolida para o tracto gastrointestinal, conduzindo assim a imprecisão na dose administrada. Outra abordagem amplamente utilizada é a instilação transtraqueal, que envolve traqueostomia sob anestesia forte para expor a traqueia e a instilação de um agente directamente no tracto respiratório. 14 No entanto, tal não só podeum procedimento ser indesejável devido ao seu invasividade, mas também consome tempo, exige um pouco de formação, e provoca uma lesão potente ao tracto respiratório têm sido desenvolvidos. 15,16 Vários protocolos que envolvem a administração directa de agentes para o traqueia, sem a necessidade de intervenção cirúrgica, 16,17,18,19,20 tempos de recuperação, mas estes métodos envolvem estendidos causada pelos anestésicos potentes, o uso de equipamento caro (isto é., otoscope / laringoscopio, disponíveis comercialmente placas de procedimento, de fibra óptica fios, etc.), um excesso de manipulação na cavidade oral, e a incerteza sobre a dosagem. Este artigo descreve um método relativamente fácil de administração por entubação que permite que um investigador de forma rápida, barata, e infundir de forma fiável um reagente para o pulmão de murino com o risco reduzido de danos residuais para os tecidos circundantes.

<table border="0" cellpadding="0" cellspacing="0" width="1528">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:481px;">Bleomycin For Injection, 30 units/vial</td>
			<td style="width:304px;">APP Pharmaceuticals, LLC</td>
			<td style="width:139px;">103720</td>
			<td style="width:604px;">For best results, BLM should be suspended in PBS, aliquoted, and stored as single use lyophilzed aliquots</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blunt End Forceps</td>
			<td style="width:304px;">N/A</td>
			<td style="width:139px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tongue Depressor (i.e. bent Valleylab Blade Electrode, 2.4&#34;)&#160;</td>
			<td style="width:304px;">Covidien</td>
			<td>E1551G</td>
			<td>Before use, create a 45 degree bend 1.5 cm&#160; from the blade tip. A suitable depressor can also be created from any metal implement of similar dimensions.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Exel Safelet Catheter 22G X 1&#34;</td>
			<td style="width:304px;">Exel International</td>
			<td>26746</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 mL Slip-tip Disposable Tuberculin Syringe (200/sp, 1600/ca)</td>
			<td style="width:304px;">BD</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.2ml Pipettor and Filter Tips</td>
			<td style="width:304px;">N/A</td>
			<td style="width:139px;">N/A</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:481px;">Fiber-Lite Illuminator. Model 181-1: Model 180 mated with Standard Dual Gooseneck illuminator</td>
			<td style="width:304px;">Dolan Jenner Industries, Inc.</td>
			<td style="width:139px;">181-1</td>
			<td>Lower output LED illuminators are not recommended as they fail to suficiently illuminate the trachea.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Intubation Board</td>
			<td style="width:304px;">N/A</td>
			<td style="width:139px;">N/A</td>
			<td>See Diagram 1.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Colored Label Tape: 0.5&#160;in. Wide</td>
			<td style="width:304px;">Fisherbrand</td>
			<td>15-901-15A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Oxygen&#160;</td>
			<td style="width:304px;">N/A</td>
			<td style="width:139px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate-Buffered Saline, 1X</td>
			<td style="width:304px;">Corning</td>
			<td>21-040-CV</td>
			<td>Product should be sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Non-Sterile Silk Black Braided Suture Spool, 91.4 m, Size 4-0</td>
			<td style="width:304px;">Harvard Apparatus</td>
			<td>517698</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Table Top Anesthesia Machine Isoflurane</td>
			<td style="width:304px;">Highland Medical Equipment</td>
			<td>N/A</td>
			<td>http://www.highlandmedical.net/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Slide Top Induction Mouse Isoflurane Chamber</td>
			<td>MIP / Anesthesia Technologies</td>
			<td>AS-01-0530-SM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FORANE (isoflurane, USP) Liquid For Inhalation 100 mL&#160;</td>
			<td style="width:304px;">Baxter</td>
			<td>1001936040</td>
			<td></td>
		</tr>
		<tr height="21">
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			<td height="21" style="height:21px;">IgM ELISA Quantification Kit&#160;</td>
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an improved method for rapid intubation of the trachea in mice

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity is a commonly used model to study both acute lung injury and fibrosis, and multiple methods have been developed for administering bleomycin (and other toxic agents) into the lungs. However, many of these approaches, such as transtracheal instillation, have inherent drawbacks, including the need for strong anesthetics and survival surgery. This paper reports a quick, reproducible method of intratracheal intubation that involves mild inhaled anesthesia, visualization of the trachea, and the use of a surrogate spirometer to confirm exposure. As a proof of concept, 8-12 week old C57BL/6 mice were administered either 2.0 U/kg of bleomycin or an equivalent volume of PBS, and both damage and fibrotic endpoints were measured post-exposure. This procedure allows researchers to treat a large cohort of mice in a relatively short period with little expense and minimal post-procedure care.

Ratos entubados foram monitorados diariamente para perda de peso e angústia, e sacrificados 4, 10 ou 17 dias depois através de injecção intraperitoneal de 2,5% 2,2,2 tribromoethanol. Lavado broncoalveolar (LBA) foi coletado em três lavagens de PBS como descrito em outra parte 21 , e o pulmão direito foi fixado em 10% de formalina, embebidos em parafina, e coradas com tricrómio de Masson pela Universidade de Washington Histologia e imagem de ...

Watch this Scientific Journal Video about An Improved Method for Rapid Intubation of the Trachea in Mice at JoVE.com

An Improved Method for Rapid Intubation of the Trachea in Mice

Um método melhorado para rápida de intubação da traqueia em Ratos

Este artigo apresenta um método rápido e simples para a administração de bleomicina diretamente na traquéia do mouse através de intubação. As principais vantagens deste método é que ele é altamente reprodutível, fácil de dominar, e não requer equipamento especializado ou tempos de recuperação longos.

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity ...

an-improved-method-for-rapid-intubation-of-the-trachea-in-mice

Research

JoVE Journal

Medicine

25.0K visualizações.  University of Washington.  Este artigo apresenta um método rápido e simples para a administração de bleomicina diretamente na traquéia do mouse através de intubação. As principais vantagens deste método é que ele é altamente reprodutível, fácil de dominar, e não requer equipamento especializado ou tempos de recuperação longos. 

Vídeo: An Improved Method for Rapid Intubation of the Trachea in Mice

Guidelines for Elective Pediatric Fiberoptic Intubation

Fiberoptic intubation in pediatric patients is often required especially in difficult airways of syndromic patients i.e. Pierre Robin Syndrome. Small babies will desaturate very quickly if ventilation is interrupted mainly to high metabolic rate. We describe guidelines to perform a safe fiberoptic intubation while maintaining spontaneous breathing throughout the procedure. Steps requiring the use of propofol pump, fentanyl, glycopyrrolate, red rubber catheter, metal insuflation hook, afrin, lubricant and lidocaine spray are shown.

We describe guidelines to perform a safe and efficient elective fiberoptic intubation in pediatric patients while maintaining spontaneous ventilation.

Diretrizes para Intubação Pediátrica Eletiva Fiberoptic

Fiberoptic intubation in pediatric patients is often required especially in difficult airways of syndromic patients i.e. Pierre Robin Syndrome. Small ...

Medicina

Orthotopic Aortic Transplantation in Mice for the Study of Vascular Disease

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. This is largely true, but there are a number of important principles that can reduce the difficulty of these procedures and enhance reproducibility. Orthotopic aortic transplantation is an excellent procedure in which to learn these principles because it involves only two end-to-end anastomoses, but requires good suturing technique and handling of the vessels for consistent success. This procedure begins with the procurement of a length of abdominal aorta from a donor animal, followed by division of the native aorta in the recipient. The procured aorta is then placed between the divided ends of the recipient aorta and sutured into place using end-to-end anastomoses. To accomplish this objective successfully requires a high degree of concentration, good tools, a steady hand, and an appreciation of how easily the vasculature of a mouse can be damaged, resulting in thrombosis. Learning these important principles is what occupies most of the beginner's time when learning microsurgery in small rodents. Throughout this protocol, we refer to these important points. This model can be used to study vascular disease in a variety of different experimental systems1-8. In the context shown here, it is most often used for the study of post-transplant vascular disease, a common long-term complication of solid organ transplantation in which intimal hyperplasia occurs within the allograft. The primary advantage of the model is that it facilitates quantitative morphometric analyses and the transplanted vessel lies contiguous to the endogenous vessel, which can serve as an additional control9. The technique shown here is most often used for mice weighing 18-25 grams. We have accumulated most of our experience using the C57BL/6J, BALB/cJ, and C3H/HeJ strains.

We describe a technique in which a section of the abdominal aorta from a mouse is transplanted orthotopically to just below the renal arteries in an allogeneic or syngeneic recipient. This technique can be useful in studies in which transplantation of large arteries of uniform size is deemed advantageous.

Transplante ortotópico de aorta em camundongos para o Estudo da Doença Vascular

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. ...

Endotracheal Intubation in Mice via Direct Laryngoscopy Using an Otoscope

Mice, both wildtype and transgenic, are the principal mammalian model in biomedical research currently. Intubation and mechanical ventilation are necessary for whole animal experiments that require surgery under deep anesthesia or measurements of lung function. Tracheostomy has been the standard for intubating the airway in these mice to allow mechanical ventilation. Orotracheal intubation has been reported but has not been successfully used in many studies because of the substantial technical difficulty or a requirement for highly specialized and expensive equipment. Here we report a technique of direct laryngoscopy using an otoscope fitted with a 2.0 mm speculum and using a 20 G&#160;intravenous catheter as an endotracheal tube. We have used this technique extensively and reliably to intubate and conduct accurate assessments of lung function in mice. This technique has proven safe, with essentially no animal loss in experienced hands. Moreover, this technique can be used for repeated studies of mice in chronic models.

Endotracheal Intubation in Mice via Direct Laryngoscopy Using an Otoscope

We have developed a simple, reliable, and relatively inexpensive method for endotracheal intubation in mice via direct laryngoscopy using an otoscope with a 2.0 mm speculum. This technique is atraumatic and can be used for repeated measurements in chronic experiments. We find it superior to tracheostomy or previously reported nonsurgical techniques.

Entubação endotraqueal em Ratos<em&gt; Via</em&gt; Laringoscopia direta Usando um otoscópio

Mice, both wildtype and transgenic, are the principal mammalian model in biomedical research currently. Intubation and mechanical ventilation are ...

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

Um método simples de intubação Lung Rato

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

Melhor método para o estabelecimento de uma In Vitro barreira hemato - encefálica modelo baseado em células endoteliais do cérebro de suínos

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

Assessment and Characterization of Hyaloid Vessels in Mice

In the eye, the embryonic hyaloid vessels nourish the developing lens and retina and regress when the retinal vessels develop. Persistent or failed regression of hyaloid vessels can be seen in diseases such as persistent hyperplastic primary vitreous (PHPV), leading to an obstructed light path and impaired visual function. Understanding the mechanisms underlying the hyaloid vessel regression may lead to new molecular insights into the vascular regression process and potential new ways to manage diseases with persistent hyaloid vessels. Here we describe the procedures for imaging hyaloid in live mice with optical coherence tomography (OCT) and fundus fluorescein angiography (FFA) and a detailed technical protocol of isolating and flat-mounting hyaloid ex vivo for quantitative analysis. Low-density lipoprotein receptor-related protein 5 (LRP5) knockout mice were used as an experimental model of persistent hyaloid vessels, to illustrate the techniques. Together, these techniques may facilitate a thorough assessment of hyaloid vessels as an experimental model of vascular regression and studies on the mechanism of persistent hyaloid vessels.

This protocol describes both in vivo and ex vivo methods to fully visualize and characterize hyaloid vessels, a model of vascular regression in mouse eyes, using optical coherence tomography and fundus fluorescein angiography for the live imaging and ex vivo isolation and subsequent flat mount of hyaloid for quantitative analysis.

Avaliação e caracterização de vasos Hialóides em camundongos

In the eye, the embryonic hyaloid vessels nourish the developing lens and retina and regress when the retinal vessels develop. Persistent or failed ...

A Revised Method for Inducing Secondary Lymphedema in the Hindlimb of Mice

Animal models are of paramount importance in the research of lymphedema in order to understand the pathophysiology of the disease but also to explore potential treatment options. This mouse model allows researchers to induce significant lymphedema lasting at least 8 weeks. Lymphedema is induced using a combination of fractioned radiotherapy and surgical ablation of lymphatics. This model requires that the mice get a dose of 10 Gray (Gy) radiation before and after surgery. The surgical part of the model involves ligation of three lymph vessels and extraction of two lymph nodes from the mouse hindlimb. Having access to microsurgical tools and a microscope is essential, due to the small anatomical structures of mice. The advantage of this model is that it results in statistically significant lymphedema, which provides a good basis for evaluating different treatment options. It is also a great and easily available option for microsurgical training. The limitation of this model is that the procedure can be time consuming, especially if not practiced in advance. The model results in objectively quantifiable lymphedema in mice, without causing severe morbidity and has been tested in three separate projects.

This animal model enables researchers to induce statistically significant secondary lymphedema in the hindlimb of mice, lasting at least 8 weeks. The model can be used to study the pathophysiology of lymphedema and to investigate novel treatment options.

Um método revisado para induzir o linfedema secundário no hindlimb dos ratos

Animal models are of paramount importance in the research of lymphedema in order to understand the pathophysiology of the disease but also to explore ...

Repeated Orotracheal Intubation in Mice

The literature describes several methods for mouse intubation that either require visualization of the glottis through the oral cavity or incision in the ventral neck for direct confirmation of cannula placement in the trachea. The relative difficulty or the tissue trauma induced to the subject by such procedures can be an impediment to an investigator&#8217;s ability to perform longitudinal studies. This article illustrates a technique in which physical manipulation of the mouse following the use of a depilatory to remove hair from the ventral neck permits transcutaneous visualization of the trachea for orotracheal intubation regardless of degree of skin pigmentation. This method is innocuous to the subject and easily achieved with a limited understanding of murine anatomy. This refined approach facilitates repeated intubation, which may be necessary for monitoring progression of disease or instillation of treatments. Using this method may result in a reduction of the number of animals and technical skill required to measure lung function in mouse models of respiratory disease.

The goal of this article is to describe a refined method of intubation of the laboratory mouse. The method is noninvasive and, therefore, ideal for studies that require serial monitoring of respiratory function and/or instillation of treatments into the lung.

Intubação Orotraqueal Repetida em Camundongos

The literature describes several methods for mouse intubation that either require visualization of the glottis through the oral cavity or incision in the ...

Semi-Minimal Invasive Method to Induce Myocardial Infarction in Rats and the Assessment of Cardiac Function by an Isolated Working Heart System

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily and highly reproducible MI induction procedure is required to obtain further insight and better understanding of the underlying pathological changes. This procedure can also be used to evaluate the effects or potency of new and promising treatments (as drugs or interventions) in acute MI, subsequent remodeling and heart failure (HF). After intubation and pre-operative preparation of the animal, an anesthetic protocol with isoflurane was performed, and the surgical procedure was conducted&#160;quickly. Using a minimally invasive approach, the left anterior descending artery (LAD) was located and occluded by a ligature. The occlusion can be performed acutely for subsequent reperfusion (ischemia/reperfusion injury). Alternatively, the vessel can be ligated permanently to investigate the development of chronic MI, remodeling or HF. Despite common pitfalls, the drop-out rates are minimal. Various treatments such as remote ischemic conditioning can be examined for their cardioprotective potential pre-, peri- and post-operatively. The post-operative recovery was quick as the anesthesia was precisely controlled and the duration of the operation was short. Post-operative analgesia was administered for three days. The minimally invasive procedure reduces the risk of infection and inflammation. Furthermore, it facilitates rapid recovery. The &#8220;working heart&#8221; measurements were performed ex vivo and enabled precise control of preload, afterload and flow. This procedure requires specific equipment and training for adequate performance. This manuscript provides a detailed step-by-step introduction for conducting these measurements.

This article presents an efficient method to perform myocardial ischemia and subsequent chronic reperfusion in rats using a minimally invasive approach. In addition, left ventricular hemodynamic function of rats is assessed by echocardiography and isolated working heart methods.&#160;

Método invasivo semi-mínimo para induzir infarto do miocárdio em ratos e a avaliação da função cardíaca por um sistema cardíaco de trabalho isolado

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily ...

A Modified Simple Method for Induction of Myocardial Infarction in Mice

Myocardial infarction (MI) represents one of the leading causes of death. MI models are widely used for investigating the pathomechanisms of post-MI remodeling and evaluation of novel therapeutics. Different methods (e.g., isoproterenol treatment, cryoinjury, coronary artery ligation, etc.) have been used to induce MI. Compared with isoproterenol treatment and cryoinjury, coronary artery ligation may better reflect the ischemic response and chronic remodeling after MI. However, traditional methods for coronary ligation in mice are technically challenging. The current study describes a simple and efficient process for induction of MI in mice with readily available materials. The mouse chest skin was cut open under stable anesthesia. The heart was immediately externalized through the intercostal space after blunt separation of the pectoralis major and pectoralis minor. The left anterior descending branch (LAD) was ligated with a 6-0 suture 3 mm from its origin. Following LAD ligation, staining with 2,3,5-Triphenyltetrazolium chloride (TTC) indicated successful induction of MI and temporal changes of post-MI scar size. Meanwhile, survival analysis results showed overt mortality within 7 days after MI, mainly due to cardiac rupture. Moreover, post-MI echocardiographic assessment demonstrated successful induction of contractile dysfunction and ventricular remodeling. Once mastered, an MI model can be established in mice within 2-3 min with readily available materials.

Under adequate anesthesia, the mouse heart was externalized through the intercostal space, and myocardial infarction was successfully induced by ligating the left anterior descending artery (LAD) using materials readily available in most laboratories.

Um Método Simples Modificado para Indução de Infarto do Miocárdio em Camundongos

Myocardial infarction (MI) represents one of the leading causes of death. MI models are widely used for investigating the pathomechanisms of post-MI ...