Name: establishment of deep hypothermic circulatory arrest in rats
Uploaded: 2022-12-16T12:30:00
Description: Watch this Scientific Journal Video about Establishment of Deep Hypothermic Circulatory Arrest in Rats at JoVE.com

Les auteurs remercient Liang Zhang d’avoir aidé à collecter les données vidéo pendant l’expérience. Cette étude a été financée par la Fondation nationale des sciences naturelles de Chine (numéro de subvention : 82070479) et le Fonds de recherche fondamentale pour les universités centrales (numéro de subvention : 3332022128).

Les protocoles ont fait l’objet d’un examen institutionnel et ont reçu l’approbation du Comité institutionnel de soin et d’utilisation des animaux, Hôpital Fuwai, Académie chinoise des sciences médicales (FW-2021-0005). Toutes les procédures expérimentales ont été effectuées conformément au Guide pour le soin et l’utilisation des animaux de laboratoire publié par les National Institutes of Health.
REMARQUE : Les rats Sprague-Dawley mâles (poids : 500-600 g, âge : 12-14 ...

<ol>
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C., Cao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Triptolide+improves+neurobehavioral+functions,+inflammation,+and+oxidative+stress+in+rats+under+deep+hypothermic+circulatory+arrest.">Triptolide improves neurobehavioral functions, inflammation, and oxidative stress in rats under deep hypothermic circulatory arrest.</a> Aging. 13 (2), 3031-3044 (2021).</li><li>Liu, 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+novel+target+to+reduce+microglial+inflammation+and+neuronal+damage+after+deep+hypothermic+circulatory+arrest.">A novel target to reduce microglial inflammation and neuronal damage after deep hypothermic circulatory arrest.</a> Journal of Thoracic and Cardiovascular Surgery. 159 (6), 2431-2444 (2020).</li><li>Pinto, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+extracellular+isoform+of+superoxide+dismutase+has+a+significant+impact+on+cardiovascular+ischaemia+and+reperfusion+injury+during+cardiopulmonary+bypass.">The extracellular isoform of superoxide dismutase has a significant impact on cardiovascular ischaemia and reperfusion injury during cardiopulmonary bypass.</a> European Journal of Cardio-Thoracic Surgery. 50 (6), 1035-1044 (2016).</li><li>Hirao, S., Masumoto, H., Itonaga, T., Minatoya, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+recovery+cardiopulmonary+bypass+model+without+transfusion+or+inotropic+agents+in+rats.">A recovery cardiopulmonary bypass model without transfusion or inotropic agents in rats.</a> Journal of Visualized Experiments. (133), e56986 (2018).</li><li>Ha, J. Y., Kim, J. S., Kim, S. E., Son, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+activation+of+mitophagy+and+autophagy+by+staurosporine+protects+against+dopaminergic+neuronal+cell+death.">Simultaneous activation of mitophagy and autophagy by staurosporine protects against dopaminergic neuronal cell death.</a> Neuroscience Letters. 561, 101-106 (2014).</li><li>Yamamoto, A., Yue, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+and+its+normal+and+pathogenic+states+in+the+brain.">Autophagy and its normal and pathogenic states in the brain.</a> Annual Review of Neuroscience. 37, 55-78 (2014).</li><li>You, X. 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C., Cao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beneficial+effects+of+chlorogenic+acid+treatment+on+neuroinflammation+after+deep+hypothermic+circulatory+arrest+may+be+mediated+through+CYLD/NF-&#954;B+signaling.">Beneficial effects of chlorogenic acid treatment on neuroinflammation after deep hypothermic circulatory arrest may be mediated through CYLD/NF-&#954;B signaling.</a> Brain Research. 1767, 147572 (2021).</li><li>Li, Y. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+profiles+of+circular+RNAs+in+the+rat+hippocampus+after+deep+hypothermic+circulatory+arrest.">Differential expression profiles of circular RNAs in the rat hippocampus after deep hypothermic circulatory arrest.</a> Artificial Organs. 45 (8), 866-880 (2021).</li><li>Linardi, D., 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=Slow+versus+fast+rewarming+after+hypothermic+circulatory+arrest:+effects+on+neuroinflammation+and+cerebral+oedema.">Slow versus fast rewarming after hypothermic circulatory arrest: effects on neuroinflammation and cerebral oedema.</a> European Journal of Cardiothoracic Surgery. 58 (4), 792-780 (2020).</li><li>Engelman, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Society+of+Thoracic+Surgeons,+The+Society+of+Cardiovascular+Anesthesiologists,+and+The+American+Society+of+ExtraCorporeal+Technology:+Clinical+practice+guidelines+for+cardiopulmonary+bypass--Temperature+management+during+cardiopulmonary+bypass.">The Society of Thoracic Surgeons, The Society of Cardiovascular Anesthesiologists, and The American Society of ExtraCorporeal Technology: Clinical practice guidelines for cardiopulmonary bypass--Temperature management during cardiopulmonary bypass.</a> Annals of Thoracic Surgery. 100 (2), 748-757 (2015).</li><li>Jenke, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AdipoRon+attenuates+inflammation+and+impairment+of+cardiac+function+associated+with+cardiopulmonary+bypass-induced+systemic+inflammatory+response+syndrome.">AdipoRon attenuates inflammation and impairment of cardiac function associated with cardiopulmonary bypass-induced systemic inflammatory response syndrome.</a> Journal of the American Heart Association. 10 (6), 018097 (2021).</li></ol>

La canulation est la procédure la plus fondamentale pour établir le DHCA chez le rat. Avant la canulation, tremper l’artère avec 0,5 mL de lidocaïne à 2 % facilitera la canulation. Après canulation, une héparinisation avec 500 UI/kg d’héparine via la veine jugulaire externe est nécessaire pour éviter la formation de microthrombus17. Nous avons constaté à plusieurs reprises que cette dose d’héparine peut atteindre l’objectif d’un temps de coagulation activé (ACT) >4...

L’arrêt circulatoire hypothermique profond (DHCA) est utilisé en chirurgie cardiaque depuis 19531. La DHCA consiste à réduire la température centrale du patient à des niveaux profondément hypothermiques (15-22 °C) avant d’interrompre globalement le flux sanguin vers le corps2. L’arrêt circulatoire peut fournir un champ opératoire relativement sans effusion de sang. L’hypothermie profonde diminue le métabolisme, en particulier dans le cerveau et le myocarde, ce qui est une méthode efficace de protection contre l’ischémie3. Le DHCA est couramment appliqué lors de chirurgies pour les cardiopathies congénitales complexes, les maladies de l’arc aortique et même les tumeurs rénales ou surrénales avec un thrombus de veine cave 4,5. Par conséquent, l’établissement de modèles animaux DHCA fournit une référence importante pour le raffinement de la procédure et la prévention des complications en milieu clinique.
Bien que des modèles puissent être établis avec des chiens6, des lapins7 et d’autres animaux, il est préférable d’utiliser des rats en raison de leur opérabilité et de leur faible coût. Le modèle de rat DHCA a été décrit pour la première fois en 2006 par Jungwirth et al.8. Il a été constaté que la durée de l’arrêt circulatoire avait un impact sur les résultats neurologiques. Depuis lors, les modèles de rats DHCA ont été largement étudiés. Il a été précisé que le DHCA pourrait provoquer un syndrome de réponse inflammatoire systémique (SIRS)9. Dans des études ultérieures, les pharmacologues ont constaté que la neuroinflammation liée au DHCA induite par SIRS pouvait être atténuée par le resvératrol10 et le triptolide11. Notre équipe a également constaté que la neuroinflammation liée au DHCA pouvait être atténuée en inhibant la protéine de liaison à l’ARN inductible par le froid12. Dans le système cardiovasculaire, la superoxyde dismutase a un effet cardioprotecteur sur les lésions d’ischémie / reperfusion (I / R) pendant DHCA13. Ces résultats ont élargi la compréhension des processus physiopathologiques liés à la DHCA et ont offert de nouvelles orientations pour améliorer les résultats de la DHCA. Cependant, les résultats concernant l’endotoxémie, le stress oxydatif et l’autophagie après DHCA ne sont pas concluants. DHCA utilise la même technologie opérationnelle que le bypass cardiopulmonaire (CPB)14, mais sa stratégie de gestion est différente et les étapes pour générer DHCA diffèrent selon les différentes équipes 8,9,10,11. La présente étude vise à fournir une méthode pour établir la procédure DHCA chez le rat.

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establishment of deep hypothermic circulatory arrest in rats

L’arrêt circulatoire hypothermique profond (DHCA) est couramment appliqué lors des chirurgies pour les cardiopathies congénitales complexes et la maladie de l’arc aortique. La présente étude vise à fournir une méthode pour établir le DHCA chez le rat. Pour évaluer l’impact du processus DHCA sur les signes vitaux, un modèle de rat-pontage cardiopulmonaire (CPB) à température normale sans arrêt circulatoire a été utilisé comme témoin. Comme prévu, le DHCA a entraîné une diminution significative de la température corporelle et de la pression artérielle moyenne. L’analyse des gaz du sang a indiqué que le DHCA augmentait les niveaux d’acide lactique mais n’influençait pas le pH sanguin et les concentrations d’hémoglobine, d’hématocrite, de Na+, de Cl−, de K+ et de glucose. De plus, par rapport aux rats CPB à température normale, les résultats de la microscopie électronique à transmission ont montré une légère augmentation des autophagosomes hippocampiques chez les rats DHCA.

En tant que groupe témoin, les rats CPB (NtCPB) à température normale sans arrêt circulatoire ont montré une pression artérielle moyenne (MAP) et une température corporelle stables pendant toute la procédure, tandis que la MAP des rats DHCA a diminué pendant l’arrêt cardiaque (p < 0,01, Figure 3A). La température des rats DHCA a chuté rapidement pendant la phase de refroidissement et s’est rétablie progressivement pendant la phase de réchauffement. Lors du sevrage ...

Watch this Scientific Journal Video about Establishment of Deep Hypothermic Circulatory Arrest in Rats at JoVE.com

Establishment of Deep Hypothermic Circulatory Arrest in Rats

Ce protocole présente l’établissement d’un arrêt circulatoire hypothermique profond chez le rat, qui peut être appliqué pour étudier le syndrome de réponse inflammatoire systémique, les lésions d’ischémie / reperfusion, le stress oxydatif, la neuroinflammation, etc.

Établissement d’un arrêt circulatoire hypothermique profond chez le rat

Deep hypothermic circulatory arrest (DHCA) is routinely applied during surgeries for complex congenital heart disease and aortic arch disease. The present ...

Établissement d’un arrêt circulatoire hypothermique profond chez le rat. Arrêt circulatoire hypothermique profond, le DHCA est couramment appliqué lors des chirurgies de cardiopathie congénitale complexe et de maladie de l’arc aortique. La présente étude vise à fournir une méthode pour établir le DHCA chez le rat.Les protocoles ont fait l’objet d’un examen institutionnel et ont reçu l’approbation du Comité institutionnel de soin et d’utilisation des animaux, Hôpital Fuwai, Académie chinoise des sciences médicales. Travaux préparatoires. Assurez-vous que les circuits CPB contiennent un réservoir modifié à partir de morphs compte-gouttes, pompe à rouleaux, échangeur de chaleur, oxygénateur à membrane, tube de raccordement et un réservoir d’eau connecté au circuit et fait 12 millilitres d’hydroxyamidon avec un millilitre d’héparine sodique et un millilitre de solution saline amorcer le circuit avec une solution d’amorçage de 40 millilitres avec la pompe à rouleaux tournant doucement.Deux, l’anesthésie et la canulation. Connectez le tube avec le ventilateur et les paramètres latéraux. Mettez une couverture chauffante électrique et la droite et faites face à la droite avec du ruban adhésif.Appliquez une pommade ophtalmique sur les yeux pour prévenir la sécheresse. Rasez les poils de la région inguinale droite, de la région cervicale droite et de la queue avec le rasoir. Ensuite, désinfectez la peau avec de l’aldéhyde et de l’alcool.Coupez la peau et la région inguinale gauche et disséquez le muscle et les tissus osseux pour exposer la veine fémorale gauche et l’artère. Séparez soigneusement l’artère. Canuler un cathéter intraveineux de calibre 22 dans l’artère fémorale gauche.Artère ligottée et cathéter avec soie. Utilisez une solution saline contenant de l’héparine pour rincer la canule afin d’éviter la coagulation. Connectez le cathéter au capteur de presse pour surveiller la pression artérielle.Coupez la peau de la queue, puis utilisez un scalpel pour couper l’espèce superficielle de l’artère de la queue afin d’exposer l’artère de la queue qui se trouve au milieu du champ chirurgical. Canuler l’artère de la queue avec le cathéter intraveineux de calibre 22. Ligate artère et cathéter avec soie.Utilisez une solution saline contenant de l’héparine pour rincer le cathéter afin d’éviter la coagulation. Coupez la peau de la jugulaire droite de la veine puis séparez le muscle et le tissu pour exposer la veine. Insérez un cathéter intraveineux multiâge maison de calibre 16 dans la jugulaire externe droite de la veine et placez-le soigneusement dans la veine cave inférieure droite ou dans l’oreillette droite.Administrer de l’héparine sodique où la veine externe droite recouvre chaque région canulée de boules humides pour éviter la contamination. Troisièmement, connectez le circuit avec le cathéter dans l’artère de la queue et maintenez la pompe à faible débit d’un à deux millilitres par minute. Ensuite, connectez le réservoir avec le cathéter dans la veine jugulaire externe droite.Assurez-vous qu’il y a toujours un taux sanguin d’environ un centimètre dans le réservoir. Allumez le réservoir d’eau et réglez d’abord la température de l’eau à 37 degrés centigrades. Une fois que la pression artérielle est stable, augmentez doucement la pompe de débit jusqu’à 80 à 100 millilitres par minute.Quatre, le refroidissement. Réglez la température ambiante à environ 20 degrés centigrades. Mettez des glaçons dans ces gants jetables, puis mettez-les sur la tête et les côtés droits.La température du réservoir a été suggérée en temps réel en fonction de la température rectale du riz. Modifier correctement les paramètres pertinents du ventilateur en fonction des résultats de l’analyse des gaz du sang. Cinq, l’arrêt circulatoire par hypothermie profonde.Ensuite, la température rectale chute à 15 à 20e centigrade. Changez le sac de glace et jetez les gants pour assurer le maintien de l’hypothermie profonde pendant l’arrêt circulatoire. Arrêtez la pompe à rouleaux.Gardez le réservoir en contact avec l’environnement et drainez lentement le sang de la veine jugulaire externe vers le réservoir. Faites attention à la réforme de la pression artérielle. Après qu’il n’y a pas de pression artérielle et de rythme cardiaque, arrêtez le drainage et gardez le réservoir fermé.Éteignez le ventilateur. Six, l’échauffement et la reperfusion. Retirez tous les gants jetables et augmentez la température ambiante à 25 degrés centigrades.Rétablir l’interventilation oxygène de la mémoire tout en maintenant l’écrêtage du tube de drainage veineux. Allumez la pompe à rouleaux pour vous assurer que le sang dans le réservoir est lentement de retour vers le bon corps. Allumez le ventilateur après que le niveau sanguin dans le réservoir reste un centimètre.Utilisez le tube de drainage et drainez lentement le sang de l’oreillette droite vers le réservoir. Allumez la lampe chauffante, le coussin chauffant et le réservoir d’eau. Réglez la température du réservoir d’eau à 25 degrés centigrades.Ensuite, réglez-le à la température de gauche en temps opportun. Retirez la lampe chauffante lorsque la température rectale est revenue à 34 degrés centigrades. Septièmement, le sevrage de la CPB.Réduire lentement et progressivement le rouleau de débit de la pompe et ajuster la vitesse du genre veine dans le débit réduire à un millilitre par minute. Gardez le réservoir en contact avec l’environnement. Infuser le sang restant dans un circuit avec un débit d’un millilitre par minute.Arrêtez l’oxygénation de la mémoire et la pompe à rouleaux. Euthanasié après une période de ventilation mécanique et une anesthésie profonde. Résultats représentatifs.Comme groupe témoin, la température normale CPB, NtCPB, rat. Sans l’arrêt circulatoire a montré une pression artérielle moyenne stable MAP et la température corporelle pendant toute la procédure. Alors que le MAP des rats DHCA a diminué pendant l’arrêt cardiaque.La température des rats DHCA a chuté rapidement pendant la phase de refroidissement et s’est rétablie progressivement pendant la phase de rotation. Le sevrage du circuit DHCA la température des rats DHCA est revenue à la normale. Les données ci-dessus indiquaient que notre procédure DHCA imitait les changements réels de température et de MAP des patients dans les cliniques.L’effet du processeur DHCA et de l’augmentation a été étudié par analyse des gaz du sang. Après le contact du sang total avec la solution primaire, la concentration d’hémoglobine était supérieure à six grammes par déciportée dans les deux groupes. Le sevrage de la surface DHCA, il est passé à neuf grammes par déciportée en raison de la réinjection du sang restant dans le circuit CPB dans la droite.L’hématocrite, HCT, a montré une tendance similaire à HB. Après la reperfusion DHCA, l’acide de lactate a augmenté rapidement, ce qui était plus prononcé dans le groupe DHCA. Le pH a diminué après la procédure DHCA, ce qui était probablement le résultat de l’accumulation d’acide lactate. Pendant toute l’expérience, la concentration de sodium, de chlorure, d’ions potassium et de glucose n’a pas montré de différences significatives à chaque point 10.Ce résultat suggère que le DHCA n’a induit que l’augmentation de l’acide lactate, mais n’a pas influencé le pH sanguin et la concentration d’hémoglobine, d’hématocrite, d’ions sodium, d’ions chlorure, d’ions potassium et de glucose. Afin d’évaluer l’impact de la DHCA sur l’autophagie neuronale, nous avons utilisé l’électromicroscope à transmission et avons étonnamment trouvé et augmenté le nombre d’autophagosomes dans l’hippocampe des rats DHCA. Les données ci-dessus indiquent que notre modèle DHCA imite les changements réels de température et de MAP des patients dans les cliniques.Ce modèle peut être appliqué pour étudier le syndrome de réponse inflammatoire systémique, la neuroinflammation, et cetera.

establishment-of-deep-hypothermic-circulatory-arrest-in-rats

Research

JoVE Journal

Immunology and Infection

Orthotopic Hind-Limb Transplantation in Rats

Composite tissue allotransplantation (CTA) now represents a valid therapeutic option after the loss of a hand, forearm or digits and has become a novel therapeutic entity in reconstructive surgery. However, long term high-dose multi-drug immunosuppressive therapy is required to ensure graft survival, bearing the risk of serious side effects which halters broader application. Further progression in this field may depend on better understanding of basic immunology and ischemia reperfusion injury in composite tissue grafts.
To date, orthotopic hind limb transplantation in rats has been the preferred rodent model for reconstructive transplantation (RT), however, it is an extremely demanding procedure that requires extraordinary microsurgical skills for reattachment of vasculature, bones, muscles and nerves.
We have introduced the vascular cuff anastomosis technique to this model, providing a rapid and reliable approach to rat hind limb transplantation. This technique simplifies and shortens the surgical procedure and enables surgeons with basic microsurgical experience to successfully perform the operation with high survival and low complication rates. The technique seems to be well suited for immunological as well as ischemia reperfusion injury (IRI) studies.

Here we describe the orthotopic rat hind-limb transplantation procedure, which seems to be the gold standard in vivo model for composite tissue allotransplantation research.

Hind-Limb orthotopique transplantation chez le rat

Composite tissue allotransplantation (CTA) now represents a valid therapeutic option after the loss of a hand, forearm or digits and has become a novel ...

Recherche

Immunologie et infection

Diagnosis of Ecto- and Endoparasites in Laboratory Rats and Mice

Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized animals. Before embarking on an examination of animals for parasites, two things should be considered. One: what use will be made of the information collected, and two: which test is the most appropriate. Knowing that animals are parasitized may be something that the facility accepts, but there is often a need to treat animals and then to determine the efficacy of treatment. Parasites may be detected in animals through various techniques, including samples taken from live or euthanized animals. Historically, the tests with the greatest diagnostic sensitivity required euthanasia of the animal, although PCR has allowed high-sensitivity testing for several types of parasite. This article demonstrates procedures for the detection of endo- and ectoparasites in mice and rats. The same procedures are applicable to other rodents, although the species of parasites found will differ.

This article describes various procedures for screening rats and mice to detect endo- or ectoparasitism. Several diagnostic assays will be demonstrated, both those suitable for use on live animals and those used after euthanasia of the animal. Photographs to aid in identification of rat and mouse parasites will be included.

Diagnostic de l&#39;ecto-et endoparasites chez les rats et les souris de laboratoire

Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized ...

Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid cell lines (LCL) in vitro. Since LCL are latently infected with EBV, they provide a model system to investigate EBV latency and virus-driven B cell proliferation and tumorigenesis1. LCL have been used to present antigens in a variety of immunologic assays2, 3. In addition, LCL can be used to generate human monoclonal antibodies4, 5 and provide a potentially unlimited source when access to primary biologic materials is limited6, 7.
A variety of methods have been described to generate LCL. Earlier methods have included the use of mitogens such as phytohemagglutinin, lipopolysaccharide8, and pokeweed mitogen9 to increase the efficiency of EBV-mediated immortalization. More recently, others have used immunosuppressive agents such as cyclosporin A to inhibit T cell-mediated killing of infected B cells7, 10-12.
The considerable length of time from EBV infection to establishment of cell lines drives the requirement for quicker and more reliable methods for EBV-driven B cell growth transformation. Using a combination of high titer EBV and an immunosuppressive agent, we are able to consistently infect, transform, and generate LCL from B cells in peripheral blood. This method uses a small amount of peripheral blood mononuclear cells that are infected in vitroclusters of cells can be demonstrated. The presence of CD23 with EBV in the presence of FK506, a T cell immunosuppressant. Traditionally, outgrowth of proliferating B cells is monitored by visualization of microscopic clusters of cells about a week after infection with EBV. Clumps of LCL can be seen by the naked eye after several weeks. We describe an assay to determine early if EBV-mediated growth transformation is successful even before microscopic clusters of cells can be demonstrated. The presence of CD23hiCD58+ cells observed as early as three days post-infection indicates a successful outcome.

We describe a method for generating transformed B cell lines using Epstein-Barr virus. We also illustrate a novel assay that can identify B cells destined to undergo transformation as early as three days after infection.

Création d&#39;Epstein-Barr Virus croissance transformées lignées lymphoblastoïdes

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

Mise en place d&#39;un<em&gt; In vitro</em&gt; Système d&#39;étudier le comportement intracellulaire de<em&gt; Candida glabrata</em&gt; En droits macrophages THP-1

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Complete Thymectomy in Adult Rats with Non-invasive Endotracheal Intubation

Thymectomy in neonatal rodents is an established and reliable procedure for immunological studies. However, in adult rats, complications of hemorrhage and pneumothorax from pleural disruption can result in a significant mortality rate. This protocol is a simple method of rat thymectomy that utilizes a mini-sternotomy and endotracheal intubation. Intubation is accomplished with a non-invasive and easily reproducible method and allows for positive pressure ventilation to prevent pneumothorax and a controlled airway that allows sufficient time for careful thymus dissection to minimize pleural disruption. A 1.5 cm sternal incision decreases contact with mediastinal vessels and pleura, while still providing full visualization of the thymus. Following exposure of the mediastinum, the thymus is removed by blunt dissection under magnification. The pleural space is then sealed by suture closure of the pre-tracheal muscles followed by the application of surgical glue. The thorax is then closed by suture closure of the sternum, followed by suture closure of the skin. All thymectomies were complete as evidenced by immunohistochemical (IHC) staining of mediastinal tissue, and absence of na&#239;ve T-cells by flow cytometry, and the procedure had a 96% survival rate. This method is suitable when complete thymectomy with minimal complications is desired for further immunological studies in athymic adult rats.

Rodent thymectomy is a valuable technique in immunological research. Here, a protocol for complete thymectomy in adult rats using a mini-sternotomy along with non-invasive intubation and positive pressure ventilation to minimize perioperative morbidity and mortality is described.

Thymectomie complète chez des rats adultes avec les non-invasive intubation endotrachéale

Thymectomy in neonatal rodents is an established and reliable procedure for immunological studies. However, in adult rats, complications of hemorrhage and ...

Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain

This study was designed to evaluate the protective effect of the Bacillus subtilis strain against complications related to heat stress. Thirty-two Sprague-Dawley rats were used in this study. Animals were orally treated twice a day for two days with B. subtilis BSB3 strain or PBS. The next day after the last treatment, each group was divided and two experimental groups (one treated with PBS and one treated with B. subtilis) were placed at 45 oC for 25 min. Two control groups stayed for 25 min at room temperature. All rats were euthanized and different parameters were analyzed in all groups. Adverse effects of heat stress are registered by the decrease of villi height and total mucosal thickness in the intestinal epithelium; translocation of bacteria from the lumen; increased vesiculation of erythrocytes and elevation of the lipopolysaccharides (LPS) level in the blood. The protective efficacy of treatment is evaluated by prevention of these side effects. The protocol was set up for the oral treatment of rats with bacteria for prevention of heat stress complications, but this protocol can be modified and used for other routes of administration and for analysis of different compounds.

Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain

We described a protocol for prevention of heat stress effects in rats by oral pre-treatment with beneficial bacteria. This protocol can be modified and used for various routes of administration and for analysis of different compounds.

Prévention de stress thermique Effets indésirables chez les rats par<em&gt; Bacillus subtilis</em&gt; Strain

This study was designed to evaluate the protective effect of the Bacillus subtilis strain against complications related to heat stress. Thirty-two ...

Stenosis of the Inferior Vena Cava: A Murine Model of Deep Vein Thrombosis

Deep vein thrombosis (DVT) and its devastating complication, pulmonary embolism, are a severe health problem with high mortality. Mechanisms of thrombus formation in veins remain obscure. Lack of mobility (e.g., after surgery or long-haul flights) is one of the main factors leading to DVT. The pathophysiological consequence of the lack of mobility is blood flow stagnation in venous valves. Here, a model is described that mimics such flow disturbance as a thrombosis-driving factor. In this model, partial flow restriction (stenosis) in the inferior vena cava (IVC) is created. Closure of about 90% of the IVC lumen for 48 h results in development of thrombi structurally similar to those in humans. The similarities are: i) most of the thrombus volume is red, i.e., consists of red blood cells and fibrin, ii) presence of a white part (lines of Zahn), iii) non-denuded endothelial monolayer, iv) elevated plasma D-Dimer levels, and v) possibility to prevent thrombosis by low molecular weight heparin. Limitations include variable size of thrombi and the fact that a certain percentage of wild-type mice (0 - 35%) may not produce a thrombus. In addition to visual observation and measurement, thrombi may be visualized by non-invasive technologies, such as ultrasonography, which allows for monitoring the dynamics of thrombus development. At shorter time points (1 - 6 h), intravital microscopy may be applied to directly observe events (e.g., recruitment of cells to the vessel wall) preceding thrombus formation. Use of this method by several teams around the world has made it possible to uncover basic mechanisms of DVT initiation and identify potential targets that might be beneficial for its prevention.

We describe here stenosis in the inferior vena cava as a murine model of deep vein thrombosis. This model recapitulates blood flow restriction, one of the major triggers of venous thrombosis in humans.

Sténose de la veine cave inférieure : un modèle murin de la thrombose veineuse profonde

Deep vein thrombosis (DVT) and its devastating complication, pulmonary embolism, are a severe health problem with high mortality. Mechanisms of thrombus ...

Deep Vein Thrombosis Induced by Stasis in Mice Monitored by High Frequency Ultrasonography

Venous thrombosis is a common condition affecting 1 - 2% of the population, with an annual incidence of 1 in 500. Venous thrombosis can lead to death through pulmonary embolism or results in the post-thrombotic syndrome, characterized by chronic leg pain, swelling, and ulceration, or in chronic pulmonary hypertension resulting in significant chronic respiratory compromise. This is the most common cardiovascular disease after myocardial infarction and ischemic stroke and is a clinical challenge for all medical disciplines, as it can complicate the course of other disorders such as cancer, systemic disease, surgery, and major trauma.
Experimental models are necessary to study these mechanisms. The stasis model induces consistent thrombus size and a quantifiable amount of thrombus. However, it is necessary to systematically ligate side branches of the inferior vena cava to avoid variability in thrombus sizes and any erroneous data interpretation. We have developed a non-invasive technique to measure thrombus size using ultrasonography. Using this technique, we can assess thrombus development and resolution over time in the same animal. This approach limits the number of mice required for quantification of venous thrombosis consistent with the principle of replacement, reduction, and refinement of animals in research. We have demonstrated that thrombus weight and histological analysis of thrombus size correlate with measurement obtained with ultrasonography. Therefore, the current study describes how to induce deep vein thrombosis in mice using the inferior vena cava stasis model and how to monitor it using high frequency ultrasound.

The present protocol describes the steps to obtain venous thrombosis using a stasis model. In addition, we are using a non-invasive method to measure thrombus formation and resolution over time.

Thrombose veineuse profonde induite par stase chez les souris surveillé par échographie de haute fréquence

Venous thrombosis is a common condition affecting 1 - 2% of the population, with an annual incidence of 1 in 500. Venous thrombosis can lead to death ...

Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

The skin is an extremely extended organ of the body and, due to this large surface, it is continuously exposed to microorganisms. Skin damage can easily lead to infections in the dermis which can, in turn, result in the dissemination of pathogens into the bloodstream. Understanding how the immune system fights infections at the very early stage and how the host can eliminate the pathogens is an important step to set the base for future therapeutic interventions. Here we describe a model of Candida albicans infection that can visualize the processes that occur early during an infection, including when the pathogen has passed the epithelial barrier, as well as the immune response elicited by the C. albicans invasion. We used this infection model to perform histological analyses that show the immune cells that infiltrate the skin as well as the presence and localization of the pathogen. Samples collected after the infection can be processed for RNA extraction.

Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

Here we describe a protocol that allows histological and molecular analysis of skin samples after Candida albicans intradermal injection. This protocol maintains the structural integrity of the skin and allows for the localization of tissue-resident or newly recruited immune cells as well as the pathogen distribution.

Profonde cutanée Injection comme un modèle d’Infection de la peau de Candida albicans pour les Analyses histologiques

The skin is an extremely extended organ of the body and, due to this large surface, it is continuously exposed to microorganisms. Skin damage can easily ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

Mise en place d’une Infection virale et analyse de l’Interaction hôte-Virus chez Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...