Les auteurs remercient Christiane Pahrmann (Lab Manager).

<ol><li> Homme souris Balb / C (8-12 semaines) sont achetés auprès de Charles River Laboratories (Sulzfeld, Allemagne). Les souris sont logées dans des conditions classiques, nourri des souris standard de la nourriture et eau ad libitum. </li><li> Isofluran 2% est utilisée pour l&#39;anesthésie. </li></ol> PRÉPARATION DES DONATEURS <ol><li> Raser les poils abdominaux et désinfecter la zone en utilisant betaisodona. </li><li> Sous vue microscopique, effectuer une incision médiane cervicale au niveau du larynx au sternum. </li><li> Retirez la graisse sous-cutanée et les muscles sangle pour obtenir une vue claire de la trachée. </li><li> Disséquer la trachée de tout les tissus environnants, comme l&#39;œsophage, les nerfs, les artères et les tissus conjonctifs. </li><li> Retirez l&#39;ensemble trachée (à partir du larynx à la bifurcation). </li><li> Rincer la greffe avec une solution saline froide et conserver la greffe à 4 ° C. </li><li> Le donneur est euthanasié par dislocation cervicale après la récolte de la trachée. </li></ol> DESTINATAIRE: transplantation hétérotopique <ol><li> Raser les poils abdominaux dans une large marge autour du site d&#39;incision et de désinfecter la zone à trois reprises en utilisant betaisodona (bétadine), suivi par l&#39;alcool. Les yeux doivent être lubrifiés avec un produit pommade ophtalmique pour éviter les cornées de s&#39;assécher. </li><li> Effectuer une laparotomie médiane et le lieu de l&#39;intestin dans un stérile, gants moistured. </li><li> Passez le grand omentum attentivement. Placez le greffon dans le centre et fixer avec une suture simple en utilisant 8-0 (Prolène, Ethicon, Allemagne). </li><li> Couvrir entièrement la greffe avec le grand omentum et fixer la greffe avec une suture simple 8-0 (Prolène, Ethicon, Allemagne). </li><li> Relocaliser les intestins dans l&#39;abdomen et rincer à chaud, une solution saline stérile avant la fermeture. </li><li> Près de 2 couches - paroi abdominale et la couche de la peau avec un motif en continu en utilisant 7-0 Prolène pour le muscle et 7-0 Vicryl pour la peau. </li></ol> DESTINATAIRE: LA TRANSPLANTATION orthotopique <ol><li> Raser les poils abdominaux dans une large marge autour du site d&#39;incision et de désinfecter la zone à trois reprises en utilisant betaisodona (bétadine), suivi par l&#39;alcool. Les yeux doivent être lubrifiés avec un produit pommade ophtalmique pour éviter les cornées de s&#39;assécher. </li><li> Diviser les muscles dragonne pour visualiser l&#39;ensemble du complexe laryngo-trachéale. </li><li> Soigneusement disséquer la trachée des tissus environnants, prendre soin de préserver les nerfs laryngé récurrent. </li><li> Diviser les trois anneaux trachée caudale du cricoïde. L&#39;animal maintient la respiration physiologique via la trachéotomie. </li><li> S&#39;assurer que le nettoyage des bords de la trachée chez le destinataire, ainsi que dans le greffon. </li><li> Le greffon est interposé entre les défauts bénéficiaires de la trachée et orienté à maintenir la polarité anatomique. </li><li> En utilisant 8-0 (Prolène, Ethicon, Allemagne) s&#39;anastomosent greffon du donneur avec le distal (médiastin) trachée. La face postérieure de l&#39;anastomose est réalisée à la mode fonctionnement continu. La face antérieure est ensuite complétée en utilisant points séparés. </li><li> Retirez toutes les sécrétions des voies aériennes. </li><li> L&#39;anastomose proximale est ensuite complétée de la même manière que celui distale. </li><li> Assurer l&#39;intégrité des voies aériennes et adéquate, la respiration spontanée. </li><li> Relocaliser les muscles et fermer la sangle de tissu sous-cutané et la couche de peau en utilisant des sutures 6-0 (Vicryl, Ethicon, Allemagne) avec le modèle continu. </li><li> Utilisez une anesthésie par injection pour le destinataire, donc l&#39;animal conserve respiration physiologique à travers la trachée. Une combinaison de 75/1/0.2 mg / kg de propofol, du fentanyl et de la médétomidine, respectivement, est utilisée pour l&#39;anesthésie ip chez la souris. </li></ol><img src="/files/ftp_upload/1437/1437Fig1.jpg" alt="Figure 1" /> Figure 1: Donateur trachée. 1A: Donateur trachée in situ après la préparation. 1B: la trachée donateurs excisées. 1C: Donateur trachée après l&#39;explantation. <img src="/files/ftp_upload/1437/1437Fig2.jpg" alt="Figure 2" /> Figure 2: Modèle hétérotopique. 2A: Le greffon est positionné au centre du grand épiploon. 2B: La greffe est fixée aux deux extrémités par une suture simple. 2C: Le greffon est enveloppé dans le grand épiploon et fixé avec une suture simple. <img src="/files/ftp_upload/1437/1437Fig3.jpg" alt="Figure 3" /> Figure 3: modèle orthotopique. 3A: Le greffon est interposé entre les bénéficiaires de la trachée et les défauts de la paroi postérieure est anastomosée à la mode fonctionnement continu. 3B: La paroi antérieure est terminée à l&#39;aide d&#39;une suture simple. <img src="/files/ftp_upload/1437/1437Fig4.jpg" alt="Figure 4" /> Figure 4: Histologie. 4A: Réformé trachée hétérotopique transplanté après 28 jours dans la sta H + EMINIER (15x). Notez l&#39;effacement de 100% luminal. 4B: Réformé orthotopique trachée transplantée après 60 jours dans la coloration H + E (15x). Maximal anéantissement atteint luminale est d&#39;environ 45%. <table border="1"><tbody><tr><td></td><td> Hétérotopique modèle de transplantation trachéale </td><td> Orthotopique modèle de transplantation trachéale </td></tr><tr><td> Avantages </td><td> + Facile à exécuter + Effacement Luminal avec une occlusion complète des voies aériennes après 28 jours + Pas d&#39;affection physique des animaux par ADO </td><td> Ventilation + physique de la greffe + Administration des médicaments inhalés possibles + Forte réactions immunologiques telles que la production d&#39;anticorps IgM alloréactives + Milieu physiologique thoraciques + Trachéale-trachéale anastomose imite le contexte clinique </td></tr><tr><td> Inconvénients </td><td> - Pas de ventilation de la trachée transplantée - Aucune évaluation des agents pathogènes inhalés possibles - Inhibition de la clairance mucociliaire et les sécrétions retenues - Microenvironnement péritonéal, au lieu du milieu thoraciques </td><td> - La formation chirurgicale nécessaire - L&#39;effacement Luminal avec l&#39;occlusion luminale app. 45% après 60 jours - Les animaux peuvent développer des symptômes de ADO </td></tr></tbody></table> Tableau 1: Avantages et inconvénients de la transplantation orthotopique et hétérotopique trachéales.

<ol>
	<li>Adams, B., Berry, G., Huang, X., Shorthouse, R., Brazelton, T., Morris, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunosuppressive+therapies+for+the+prevention+and+treatment+of+obliterative+airway+disease+in+heterotopic+rat+trachea+allografts.">Immunosuppressive therapies for the prevention and treatment of obliterative airway disease in heterotopic rat trachea allografts.</a> Transplantation. 69, 2260-2266 .</li><li>Adams, B., Brazelton, T., Berry, G., Morris, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+respiratory+epithelium+in+a+rat+model+of+obliterative+airway+disease.">The role of respiratory epithelium in a rat model of obliterative airway disease.</a> Transplantation. 69, 661-665 .</li><li>Deuse, T., Schrepfer, S., Reichenspurner, H., Hoyt, G., Fischbein, M., Robbins, R., Pelletier, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+for+experimental+heterotopic+and+orthotopic+tracheal+transplantations+&amp;#x2013;+when+to+use+which+model;+Transplant+Immunology+17.">Techniques for experimental heterotopic and orthotopic tracheal transplantations &amp;#x2013; when to use which model; Transplant Immunology 17.</a> , 255-261 (2007).</li><li>Hele, D., Yacoub, M., Belvisi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+heterotopic+tracheal+allograft+as+an+animal+model+of+obliterative+bronchiolitis.">The heterotopic tracheal allograft as an animal model of obliterative bronchiolitis.</a> Respiratory Research. 2, 169-183 .</li><li>Hertz, M., Jessurun, J., King, M., Savic, S., Murray, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproduction+of+the+obliterative+bronchiolitis+lesion+after+heterotopic+transplantation+of+mouse+airways.">Reproduction of the obliterative bronchiolitis lesion after heterotopic transplantation of mouse airways.</a> American Journal of Pathology. 142, (1993).</li><li>McDyer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+and+murine+obliterative+bronchiolitis+in+transplant.">Human and murine obliterative bronchiolitis in transplant.</a> Proceedings of the American Thoracic Society. 4, 37-43 .</li><li>Schrepfer, S., Deuse, T., Hoyt, G., Sheikh, A., Hoffmann, J., Reichenspurner, H., Robbins, R., Pelletier, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+orthotopic+tracheal+transplantation:+the+stanford+technique.">Experimental orthotopic tracheal transplantation: the stanford technique.</a> Microsurgery. 27, 187-189 (2007).</li></ol>

Sonja Schrepfer a reçu une subvention de recherche de la Deutsche Forschungsgemeinschaft (DFG) (SCHR992/3-1).] Tous les animaux ont reçu des soins humains en conformité avec les Principes de protection des animaux de laboratoire, des expériences sur les animaux ont été effectuées en conformité avec les directives et règlements établis par la Société nationale pour la recherche médicale et le Guide pour les soins et l&#39;utilisation des animaux de laboratoire publié par le National Institutes of Health (Instituts nationaux de santé de publication 85-23, révisée en 1985). Tous les animaux ont été obtenus à partir de Charles River Laboratories (Sulzfeld, Allemagne) et ont été maintenus dans les installations de soins aux animaux de l&#39;Hôpital universitaire Eppendorf à Hambourg. Les animaux ont reçu la norme chow et eau ad libitum.

Les souris sont disponibles en différentes transgeneic et le modèle KO, et convient donc d&#39;étudier des questions liées aux mécanistes ADO (4). Les deux modèles de la transplantation trachéale montré dans cette vidéo peut être utilisé comme modèles fiables pour étudier le développement ADO. Cependant, chaque modèle démontre les avantages et les limites. La transplantation hétérotopique trachéale est facile à réaliser et ne nécessite pas...

heterotopic and orthotopic tracheal transplantation in mice used as models to study the development of  obliterative airway disease

Oblitérante des voies respiratoires maladie (OAD) est la complication majeure après transplantation pulmonaire qui limite la survie à long terme (1-7). Pour étudier la physiopathologie, le traitement et la prévention des OAD, différents modèles animaux de la transplantation de la trachée chez les rongeurs ont été développés (1-7). Ici, nous utilisons deux modèles établis de la trachée de transplantation, le modèle orthotopique et hétérotopique et démontrer leurs avantages et leurs limites. Pour le modèle hétérotopique, la trachée donneur est enveloppé dans le grand épiploon du destinataire, alors que la trachée donateurs est anastomosée à la fin-en-bout anastomose dans le modèle orthotopique. Dans les deux modèles, le développement des lésions histologiques oblitérante similaires à ADO clinique a été démontrée (1-7). Cette vidéo montre comment effectuer les deux, le hétérotopique ainsi que la technique de transplantation orthotopique de la trachée chez des souris, et compare le cours du temps de développement ADO dans les deux modèles en utilisant l&#39;histologie.

Watch this Scientific Journal Video about Heterotopic and Orthotopic Tracheal Transplantation in Mice used as Models to Study the Development of  Obliterative Airway Disease at JoVE.com

Heterotopic and Orthotopic Tracheal Transplantation in Mice used as Models to Study the Development of  Obliterative Airway Disease

Cette vidéo montre et compare deux modèles expérimentaux pour étudier le développement de maladies des voies respiratoires oblitérante (OAD) chez la souris, le modèle orthotopique transplantation hétérotopique et la trachée.

Hétérotopique et orthotopique transplantation trachéale chez des souris utilisées comme modèles pour étudier le développement de maladies des voies respiratoires oblitérante

Obliterative airway disease (OAD) is the major complication after lung transplantations that limits long term survival (1-7).
To study the ...

heterotopic-orthotopic-tracheal-transplantation-mice-used-as-models

Research

JoVE Journal

Biology

13.8K vues.  University Heart Center Hamburg. Cette vidéo montre et compare deux modèles expérimentaux pour étudier le développement de maladies des voies respiratoires oblitérante (OAD) chez la souris, le modèle orthotopique transplantation hétérotopique et la trachée.

Vidéo: Heterotopic and Orthotopic Tracheal Transplantation in Mice used as Models to Study the Development of  Obliterative Airway Disease

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Test sensibilité visuelle à la vitesse et la direction du mouvement chez les lézards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Recherche

Biologie

Heterotopic Heart Transplantation in Mice

The mouse heterotopic heart transplantation has been used widely since it was introduced by Drs. Corry and Russell in 1973. It is particularly valuable for studying rejection and immune response now that newer transgenic and gene knockout mice are available, and a large number of immunologic reagents have been developed. The heart transplant model is less stringent than the skin transplant models, although technically more challenging. We have developed a modified technique and have completed over 1000 successful cases of heterotopic heart transplantation in mice. When making anastomosis of the ascending aorta and abdominal aorta, two stay sutures are placed at the proximal and distal apexes of recipient abdominal aorta with the donor s ascending aorta, then using 11-0 suture for anastomosis on both side of aorta with continuing sutures. The stay sutures make the anastomosis easier and 11-0 is an ideal suture size to avoid bleeding and thrombosis.
When making anastomosis of pulmonary artery 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 pulmonary artery. The left wall of the inferior vena cava and donor s pulmonary artery is closed with continuing sutures in the inside of the inferior vena cava after, one knot with the proximal apex stay suture the right wall of the inferior vena cava and the donor s pulmonary artery 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 heterotopic heart transplantation model has been proven by many investigators to be an important method for studying mechanisms of rejection and immune response. However, the techniques involved are still challenging. By modifying standard techniques we have had success with more than 1000 transplants.

Transplantation cardiaque hétérotopique chez la souris

The mouse heterotopic heart transplantation has been used widely since it was introduced by Drs. Corry and Russell in 1973. It is particularly valuable ...

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &amp;#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

Quantification des γH2AX Foci en réponse aux radiations ionisantes

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Manual Restraint and Common Compound Administration Routes in Mice and Rats

Being able to safely and effectively restrain mice and rats is an important part of conducting research. Working confidently and humanely with mice and rats requires a basic competency in handling and restraint methods. This article will present the basic principles required to safely handle animals. One-handed, two-handed, and restraint with specially designed restraint objects will be illustrated. Often, another part of the research or testing use of animals is the effective administration of compounds to mice and rats. Although there are a large number of possible administration routes (limited only by the size and organs of the animal), most are not used regularly in research. This video will illustrate several of the more common routes, including intravenous, intramuscular, subcutaneous, and oral gavage. The goal of this article is to expose a viewer unfamiliar with these techniques to basic restraint and substance administration routes. This video does not replace required hands-on training at your facility, but is meant to augment and supplement that training.

Working safely and humanely with research rodents requires a core competency in handling and restraint methods. This article will present the basic principles required to safely handle and effectively administer compounds to mice and rats.

Manuel de retenue et communes voies d&#39;administration composés chez la souris et le rat

Being able to safely and effectively restrain mice and rats is an important part of conducting research.  Working confidently and humanely with mice and ...

Evaluation of Mammary Gland Development and Function in Mouse Models

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves composed of a number of terminal duct lobular units made of secretory alveoli and converging ducts1. In mice, a similar architecture is observed at pregnancy in which ducts and alveoli are interspersed within the connective tissue stroma. The mouse mammary gland epithelium is a tree like system of ducts composed of two layers of cells, an inner layer of luminal cells surrounded by an outer layer of myoepithelial cells denoted by the confines of a basement membrane2. At birth, only a rudimental ductal tree is present, composed of a primary duct and 15-20 branches. Branch elongation and amplification start at the beginning of puberty, around 4 weeks old, under the influence of hormones3,4,5. At 10 weeks, most of the stroma is invaded by a complex system of ducts that will undergo cycles of branching and regression in each estrous cycle until pregnancy2. At the onset of pregnancy, a second phase of development begins, with the proliferation and differentiation of the epithelium to form grape-shaped milk secretory structures called alveoli6,7. Following parturition and throughout lactation, milk is produced by luminal secretory cells and stored within the lumen of alveoli. Oxytocin release, stimulated by a neural reflex induced by suckling of pups, induces synchronized contractions of the myoepithelial cells around the alveoli and along the ducts, allowing milk to be transported through the ducts to the nipple where it becomes available to the pups 8. Mammary gland development, differentiation and function are tightly orchestrated and require, not only interactions between the stroma and the epithelium, but also between myoepithelial and luminal cells within the epithelium9,10,11. Thereby, mutations in many genes implicated in these interactions may impair either ductal elongation during puberty or alveoli formation during early pregnancy, differentiation during late pregnancy and secretory activation leading to lactation12,13. In this article, we describe how to dissect mouse mammary glands and assess their development using whole mounts. We also demonstrate how to evaluate myoepithelial contractions and milk ejection using an ex-vivo oxytocin-based functional assay. The effect of a gene mutation on mammary gland development and function can thus be determined in situ by performing these two techniques in mutant and wild-type control mice.

This method describes how to dissect and assess mammary gland development and function from mice. Excised mammary glands are assessed for the degree of development using whole mount while milk ejection is evaluated using an oxytocin-based myoepithelial cell contraction assay.

Evaluation de développement des glandes mammaires et fonction dans des modèles murins

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves ...

Diagnostic Necropsy and Selected Tissue and Sample Collection in Rats and Mice

There are multiple sample types that may be collected from a euthanized animal in order to help diagnose or discover infectious agents in an animal colony. Proper collection of tissues for further histological processing can impact the quality of testing results. This article describes the conduct of a basic gross examination including identification of heart, liver, lungs, kidneys, and spleen, as well as how to collect those organs. Additionally four of the more difficult tissue/sample collection techniques are demonstrated. Lung collection and perfusion can be particularly challenging as the tissue needs to be properly inflated with a fixative in order for inside of the tissue to fix properly and to enable thorough histologic evaluation. This article demonstrates the step by step technique to remove the lung and inflate it with fixative in order to achieve optimal fixation of the tissue within 24 hours. Brain collection can be similarly challenging as the tissue is soft and easily damaged. This article demonstrates the step by step technique to expose and remove the brain from the skull with minimal damage to the tissue. The mesenteric lymph node is a good sample type in which to detect many common infectious agents as enteric viruses persist longer in the lymph node than they are shed in feces. This article demonstrates the step by step procedure for locating and aseptically removing the mesenteric lymph node. Finally, identification of infectious agents of the respiratory tract may be performed by bacterial culture or PCR testing of nasal and/or bronchial fluid aspirates taken at necropsy. This procedure describes obtaining and preparing the respiratory aspirate sample for bacterial culture and PCR testing.

This article describes the procedures for conducting a basic postmortem examination of a mouse or rat, and the collection of basic organs, as well as more challenging sample types from for histological, microbiological, and PCR evaluation.

Autopsie de diagnostic et de certains tissus et de collecte des échantillons dans les rats et les souris

There are multiple sample types that may be collected from a euthanized animal in order to help diagnose or discover infectious agents in an animal ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Do-It-Yourself dispositif de récupération d&#39;échantillons cryoconservés tombé accidentellement dans les réservoirs de stockage cryogéniques

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Utilisation de la séquence d&#39;arrêt SecM comme un outil pour isoler les polypeptides Ribosome Bound

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Tissue Triage and Freezing for Models of Skeletal Muscle Disease

Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal results from functional, cellular, molecular, and pathological evaluations. Due to the subtlety of some pathological abnormalities seen in congenital muscle disorders and the potential for fixation to interfere with the recognition of these features, pathological evaluation of frozen muscle is preferable to fixed muscle when evaluating skeletal muscle for congenital muscle disease. Additionally, the potential to produce severe freezing artifacts in muscle requires specific precautions when freezing skeletal muscle for histological examination that are not commonly used when freezing other tissues. This manuscript describes a protocol for rapid freezing of skeletal muscle using isopentane (2-methylbutane) cooled with liquid nitrogen to preserve optimal skeletal muscle morphology. This procedure is also effective for freezing tissue intended for genetic or protein expression studies. Furthermore, we have integrated our freezing protocol into a broader procedure that also describes preferred methods for the short term triage of tissue for (1) single fiber functional studies and (2) myoblast cell culture, with a focus on the minimum effort necessary to collect tissue and transport it to specialized research or reference labs to complete these studies. Overall, this manuscript provides an outline of how fresh tissue can be effectively distributed for a variety of phenotypic studies and thereby provides standard operating procedures (SOPs) for pathological studies related to congenital muscle disease.

The analysis of skeletal muscle tissues to determine structural, functional, and biochemical properties is greatly facilitated by appropriate preparation. This protocol describes appropriate methods to prepare skeletal muscle tissue for a broad range of phenotyping studies.

Triage de tissus et de congélation pour les modèles de la maladie de muscle squelettique

Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal ...