<p class="jove_content"&gt; Les études actuelles sont soutenues par National Heart, Lung, and Blood Institute Grant R01-HL0921, R01-DK083385 et R01HL098294 à HK Eltzschig, le 1K08HL102267-01 à T. Eckle, et la Fondation pour l&#39;éducation d&#39;anesthésie et de subventions de recherche à T. Eckle et HK Eltzschig, et l&#39;American Heart Association à T. Grant Eckle et HK Eltzschig et Deutsche Forschungsgemeinschaft bourse de recherche (DFG) à M. Koeppen.</p

Remarques générales: Toutes les opérations doivent être effectuées sous un microscope droit dissection (Olympus, SZX10 avec Postes Crank Z-Axis avec STU2 perche Stand) et en utilisant un coagulateur chirurgical (11). Les groupes expérimentaux doivent correspondre le mieux possible dans l&#39;âge et le poids pour assurer la comparabilité des résultats. L&#39;administration de la température, la pression sanguine, l&#39;anesthésie et le liquide doit être stable tout au long. 1. L&#39;anesthésie et la préparation trachée <ol><li> Utilisez des souris C57BL / 6 qui sont au moins 10 semaines et avoir un corps de 22-25 g. Induire une anesthésie à l&#39;aide de pentobarbital de sodium à une dose de 70 mg / kg de poids corporel ip (6). Maintenir une anesthésie d&#39;environ de 10 mg / kg / h de pentobarbital de sodium. Soyez prudent avec le surdosage car cela pourrait considérablement baisser la pression artérielle. Re-dosage du pentobarbital - même après-heures peut conduire à une augmentation sévère du taux plasmatique. </li><li> Après l&#39;induction anesthésique, souris sécurisé en décubitus dorsal avec les membres supérieurs et inférieurs attachés à la table à l&#39;aide d&#39;un ruban et une suture attachée aux chevilles. Faites de même pour la tête en utilisant les dents. Retenue suffisant est important pour une intubation réussie et une chirurgie bien contrôlé. Avant la chirurgie, la couverture de la souris avec de l&#39;huile minérale pour réduire le risque d&#39;allergie poils de souris. Afin de s&#39;assurer que la température du corps reste stable couvrir la souris avec une pellicule de la nourriture disponible dans le commerce. </li><li> Souris sur une table chauffée à température contrôlée (RT, Effenberg, Munich, Allemagne) avec une sonde thermomètre rectal attachés au contrôleur de rétroaction thermique pour maintenir la température corporelle à 37 ° C. </li><li> Exposer la trachée chirurgicalement. Disséquer faces latérales et dorsale de la trachée du tissu conjonctif et placer deux 3,0 suture de soie chirurgicale (Harvard Apparatus, USA) à chaque 10 cm de long au-dessous de la trachée. Les sutures doivent être d&#39;environ 1 cm d&#39;intervalle. </li><li> Soigneusement inciser la trachée 3 - 4 mm en dessous du larynx entre les deux cartilages circulaire en utilisant une paire de ciseaux McPherson Vannas (8 cm, lame droite; Instruments de précision Mondiale, USA). Veillez à ne pas provoquer une hémorragie, car cela pourrait confondre les paramètres de résultats. </li><li> Effectuer une intubation trachéale avec un polyéthylène émoussée canules (Insyte 22g, Beckton Dickinson, USA). Insérez l&#39;extrémité de la canule de polyéthylène dans un angle 85 degrés dans la trachée. Puis inclinez le canules il est donc en ligne avec la lumière trachéale. Lentement l&#39;avance le tube plus bas de la trachée jusqu&#39;au bout des canules a disparu dans l&#39;ouverture du thorax. Fixer le tube dans cette position avec la suture de soie deux chirurgicale placé dorsale de la trachée (voir 1.4). </li></ol> 2. Technique des lésions pulmonaires induites par un ventilateur <ol><li> Connecter le tube d&#39;un ventilateur. Pour induire des lésions pulmonaires, nous utilisons une technique de ventilation à pression contrôlée en utilisant un servo 900 C de Siemens (DRE vétérinaire, USA). Les animaux seront ventilés en utilisant pic de pression inspiratoire de 45 mbar, la fréquence de 80 cycles / min et une pression expiratoire positive de 0-3 mbar avec une FiO 2 = 1,0. L&#39;inspiration ratio d&#39;expiration doit être de 1:1. Malgré le fait que le servo 900 C est construit comme un ventilateur pour les humains, son utilisation dans un cadre ventilateur à pression contrôlée excellents ouvrages pour la ventilation de la souris. </li><li> Moniteur de fréquence cardiaque avec un ECG (par exemple, Hewlett Packard, B blingen, Allemagne). Assurez-vous que la fréquence cardiaque ne chute pas en dessous de 400. On devrait voir un changement de l&#39;axe du cœur vers la droite, lorsque la ventilation mécanique est instituée comme un signe d&#39;augmentation de la pression artérielle pulmonaire suite à une augmentation de la pression intrathoracique. Si la souris se développe une bradycardie, vérifiez la température et la dose d&#39;anesthésique / concentration. Xylacin / kétamine anesthésie induit un cœur plein cœur de 250 / min et n&#39;est donc pas recommandé. </li><li> Appliquer une réhydratation correcte. Une infusion avec une solution saline normale 0,1 ml / heure par l&#39;intermédiaire d&#39;un cathéter artériel ou veineux doit être effectuée avant la ventilation pour assurer le dépôt veineuse suffisante. En raison des pressions de ventilation élevées le retour veineux vers le cœur est altérée, ce qui pourrait conduire à une baisse critique de la pression artérielle moyenne. En outre, un bolus salins de 500 ul pourrait être donnée ip avant la chirurgie. </li><li> Placer un cathéter dans l&#39;artère carotide pour l&#39;enregistrement continu de la pression artérielle (27). Fixer le bras au corps avant de commencer la dissection de l&#39;artère. L&#39;artère carotide est exposé via dissection des muscles paratrachéal. Suite à une exposition plus loin et d&#39;éviter soigneusement tout traumatisme tissulaire (notamment du nerf vagal), un cathéter est introduit dans le vaisseau en utilisant deux points de suture et une petite pince (37). Cela permettra d&#39;exposer un segment plus long de l&#39;artère. Placez une ligature à l&#39;extrémité de l&#39;artère carotide. Attacher une grande pince à l&#39;extrémité de la suture d&#39;obtenir ème tension ou fixere de suture à la table avec du ruban. Placez un autre fil de suture autour de l&#39;artère et de disséquer l&#39;artère à la fin distale. Ici, place une petite pince. Utilisez des ciseaux pour couper les micro une petite ouverture en diagonale dans l&#39;artère. Tenir l&#39;ouverture avec une pince fine (Dumont, WPI) et de faire progresser le cathéter bonne taille avec vos mains / forceps. Faites un noeud à votre deuxième suture et sécuriser l&#39;artère. Desserrer le collier et de faire avancer le cathéter supplémentaire. Fixez le cathéter avec plusieurs noeuds et ruban adhésif. Alternativement, le cathéter dans l&#39;artère carotide peut être lieu à la fin de l&#39;expérience pour recueillir des échantillons de sang artériel pour analyse des gaz sanguins. </li></ol> 3. Récupération des échantillons de tissus Après 3 heures de ventilation mécanique, les échantillons sont prélevés pour évaluer l&#39;étendue des lésions pulmonaires. Nous recommandons la collecte du liquide de lavage brochnoalveolar (BAL), le sang artériel et les tissus pulmonaires. <ol><li> Obtenir le liquide de LBA à la fin de l&#39;expérience. Après l&#39;approfondissement de l&#39;anesthésie, rincer la sonde trachéale avec 1 ml de phosphate saline tamponnée (PBS). Le liquide doit rester dans la trachée / poumon pendant trois secondes avant qu&#39;il ne soit reprise par la seringue connectée. Le BAL est enfichable congelés dans l&#39;azote liquide et conservés à -80 ° C pour analyse ultérieure. Soyez conscient que le volume récupéré pourrait être sensiblement inférieure à 1 ml. </li><li> Effectuer une analyse des gaz du sang à la fin de l&#39;expérience. Pour ce faire, une incision doit être faite juste en dessous du sternum. Tenez le sternum avec une pince et l&#39;étendue de l&#39;incision le long de la cage thoracique. Ensuite, le diaphragme est incisé sur les bords et est découpé dans les côtes. Maintenant, il ya une vue imprenable sur l&#39;ouverture inférieure du thorax. Soulevez le sternum avec une pince et d&#39;ouvrir le thorax par des coupures longues sur le côté droit et gauche (comme latéral que possible) afin que la paroi thoracique antérieure complète est monté-pupilles. Cela devrait être fait, alors que l&#39;animal est encore expérimentale ventilés mécaniquement. Le ventricule gauche est percé en utilisant une aiguille de 27 G ½ et l&#39;analyse du sang artériel est réalisée en utilisant le système i-STAT (Abbott, USA). Si l&#39;analyse du sang artériel de gaz doit être effectuée, un BAL ne peut être obtenu, puisque ce sera un facteur de confusion important pour les résultats. </li><li> Alternativement à la méthode ci-dessus, des échantillons de sang artériel peut être collectées via le cathéter dans l&#39;artère carotide. Cependant cela doit être fait avant la BAL est obtenu. </li><li> D&#39;accise dans les poumons en bloc en tirant sur le cœur et couper la trachée. Avoir un morceau de tissu prêt à absorber le sang, de sorte que le site chirurgical est visible. Tirez sur le coeur dans le sens de l&#39;abdomen et soigneusement couper le long de la colonne vertébrale de mobiliser tous les organes thoraciques. Coupez l&#39;aorte, enlever les organes thoraciques et les placer sur une table propre chirurgicale. </li><li> Coupez le cœur et le vaisseau principal de l&#39;échantillon de tissu. Assurez-vous qu&#39;il n&#39;ya pas de tissu thymique encore attaché au poumon. Séparez les poumons avec un ciseau et le placer dans des tubes individuels et snap-gel. Conserver à -80 ° C pour analyse ultérieure. </li></ol> 4. Mesure des lésions pulmonaires Nous vous recommandons d&#39;utiliser les paramètres résultats suivants pour évaluer l&#39;étendue des lésions pulmonaires: Effectuer une albumine ELISA (Bethyl Laboratories, USA) et un myéloperoxydase (MPO) ELISA (Hycult biotechnologie, USA) pour évaluer l&#39;ampleur du dysfonctionnement de la barrière et le montant des maladies inflammatoires cellules dans le liquide de LBA. Effectuer une MPO ELISA également former le tissu pulmonaire. Si humides à sèches ratio est d&#39;être mesurée, nous n&#39;obtenons pas le liquide de LBA et de la circulation pulmonaire n&#39;est pas rincé (voir 3.3). Mesurer le poids des poumons après l&#39;excision. Puis poumons sont lyophilisés pendant 48 h et le tissu pulmonaire est à nouveau mesurée. Puis le rapport humides à sec est mesuré en mg d&#39;eau par mg de tissu sec (5). <img src="/files/ftp_upload/2525/2525fig1.jpg" alt="Figure 1" /> Figure 1. Teneur en protéines dans la BAL en réponse à l&#39;IVL. Des souris ont été anesthésiés avec du pentobarbital, la ventilation mécanique a été institué et les souris ont été ventilés en utilisant la pression contrôlée par les paramètres (pression inspiratoire de 45 mbar, positive en fin d&#39;exspiratory pression 3 mbar, 100% d&#39;inspiration concentration en oxygène). Après 0, 1 heure, 2 et 3 de la ventilation BAL a été récoltée et la teneur en protéines a été quantifiée en utilisant un dosage acide bicinchoninique (test BCA). La variation relative de la teneur en protéines est montré normalisée à 0 heures de ventilation (n = 4 par groupe, * indique p &lt;0,05 par rapport au témoin, moyenne ± SEM)

<ol>
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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+deletion+of+the+A1+adenosine+receptor+abolishes+heart-rate+slowing+effects+of+intravascular+adenosine+in+vivo.">Selective deletion of the A1 adenosine receptor abolishes heart-rate slowing effects of intravascular adenosine in vivo.</a> PLoS One. 4, e6784-e6784 (2009).</li><li>Kohler, D., Eckle, T., Faigle, M., Grenz, A., Mittelbronn, M., Laucher, S., Hart, M. L., Robson, S. C., Muller, C. E., Eltzschig, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD39/ectonucleoside+triphosphate+diphosphohydrolase+1+provides+myocardial+protection+during+cardiac+ischemia/reperfusion+injury.">CD39/ectonucleoside triphosphate diphosphohydrolase 1 provides myocardial protection during cardiac ischemia/reperfusion injury.</a> Circulation. 116, 1784-1794 (2007).</li><li>Kuhlicke, J., Frick, J. S., Morote-Garcia, J. C., Rosenberger, P., Eltzschig, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+Inducible+Factor+(HIF)-1+Coordinates+Induction+of+Toll-Like+Receptors+TLR2+and+TLR6+during+Hypoxia.">Hypoxia Inducible Factor (HIF)-1 Coordinates Induction of Toll-Like Receptors TLR2 and TLR6 during Hypoxia.</a> PLoS ONE. 2, e1364-e1364 (2007).</li><li>Morote-Garcia, J. C., Rosenberger, P., Kuhlicke, J., Eltzschig, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIF-1-dependent+repression+of+adenosine+kinase+attenuates+hypoxia-induced+vascular+leak.">HIF-1-dependent repression of adenosine kinase attenuates hypoxia-induced vascular leak.</a> Blood. 111, 5571-5580 (2008).</li><li>Morote-Garcia, J. C., Rosenberger, P., Nivillac, N. M., Coe, I. R., Eltzschig, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia-inducible+factor-dependent+repression+of+equilibrative+nucleoside+transporter+2+attenuates+mucosal+inflammation+during+intestinal+hypoxia.">Hypoxia-inducible factor-dependent repression of equilibrative nucleoside transporter 2 attenuates mucosal inflammation during intestinal hypoxia.</a> Gastroenterology. 136, 607-618 (2009).</li><li>Reutershan, J., Vollmer, I., Stark, S., Wagner, R., Ngamsri, K. C., Eltzschig, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenosine+and+inflammation:+CD39+and+CD73+are+critical+mediators+in+LPS-induced+PMN+trafficking+into+the+lungs.">Adenosine and inflammation: CD39 and CD73 are critical mediators in LPS-induced PMN trafficking into the lungs.</a> FASEB J. 23, 473-482 (2009).</li><li>Schingnitz, U., Hartmann, K., Macmanus, C. F., Eckle, T., Zug, S., Colgan, S. P., Eltzschig, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+through+the+A2B+adenosine+receptor+dampens+endotoxin-induced+acute+lung+injury.">Signaling through the A2B adenosine receptor dampens endotoxin-induced acute lung injury.</a> J Immunol. 184, 5271-5279 (2010).</li><li>Thompson, L. F., Eltzschig, H. K., Ibla, J. C., Van De Wiele, C. J., Resta, R., Morote-Garcia, J. C., Colgan, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crucial+role+for+ecto-5'-nucleotidase+(CD73)+in+vascular+leakage+during+hypoxia.">Crucial role for ecto-5'-nucleotidase (CD73) in vascular leakage during hypoxia.</a> J. Exp. Med. 200, 1395-1405 (2004).</li><li>Aherne, C. M., Kewley, E. M., Eltzschig, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+resurgence+of+A2B+adenosine+receptor+signaling.">The resurgence of A2B adenosine receptor signaling.</a> Biochim Biophys Acta. , (2010).</li><li>Eckle, T., Koeppen, M., Eltzschig, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+extracellular+adenosine+in+acute+lung+injury.">Role of extracellular adenosine in acute lung injury.</a> Physiology (Bethesda). 24, 298-306 (2009).</li><li>Koeppen, M., Feil, R., Siegl, D., Feil, S., Hofmann, F., Pohl, U., de Wit, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=cGMP-dependent+protein+kinase+mediates+NO-+but+not+acetylcholine-induced+dilations+in+resistance+vessels+in+vivo.">cGMP-dependent protein kinase mediates NO- but not acetylcholine-induced dilations in resistance vessels in vivo.</a> Hypertension. 44, 952-955 (2004).</li></ol>

La présente étude décrit une technique d&#39;effectuer une lésion pulmonaire ventilation induite chez la souris. Ce modèle démontre blessures hautement reproductible grâce à la ventilation à haute pression. Les enquêteurs qui considèrent l&#39;étude des lésions aiguës du poumon chez les souris peuvent bénéficier de ce modèle.

<table border="1"><tbody><tr><th> Nom du réactif </th><th> Société </th><th> Numéro de catalogue </th><th> Commentaires (optionnel) </th></tr><tr><td> Pentobarbital sodique (Fatal Plus) </td><td> Ls Vortech pharmaceutique, Ltd, </td><td> VPL 9372 </td><td> 4mg/mL dans une solution saline </td></tr><tr><td> Insyte 22 G </td><td> Beckton Dickinson </td><td> n / a </td><td></td></tr><tr><td> Suture, soie 4,0 </td><td> Harvard Apparatus </td><td> 517698 </td><td></td></tr><tr><td> Suture, Prolène 8.0 </td><td> Ethicon, USA </td><td> M8739 </td><td> réutilisables </td></tr><tr><td> Siemens 900 ° C </td><td> DRE vétérinaire, USA </td><td> # 336 </td><td> rénové </td></tr><tr><td> microscope à dissection (SZX10) </td><td> Olympus </td><td> n / a </td><td> considère la distance de travail généreuse </td></tr><tr><td> Tableau de chauffage </td><td> Rt, Effenberger, Allemagne </td><td> n / a </td><td> fournisseur de la seule et unique </td></tr><tr><td> Dispositif de pression artérielle </td><td> Sens Cyber, Inc </td><td> BPM02 </td><td></td></tr><tr><td> STAT-je </td><td> Abbott </td><td> n / a </td><td></td></tr></tbody></table>

pressure controlled ventilation to induce acute lung injury in mice

Les modèles murins sont largement utilisées pour étudier les lésions aiguës de systèmes différents organes (1-34). Lésion pulmonaire aiguë (ALI), qui se produit avec une ventilation mécanique prolongée, contribue à la morbidité et la mortalité des maladies graves, et des études sur de nouvelles cibles génétiques ou pharmacologiques sont des zones d&#39;intense investigation (1-3, 5, 8, 26, 30, 33 -36). ALI est défini par la survenue aiguë de la maladie, ce qui conduit à la non-cardiaques œdème pulmonaire et une insuffisance pulmonaire ultérieure de l&#39;échange de gaz (36). Nous avons développé un modèle murin de l&#39;ALI en utilisant une ventilation à pression contrôlée pour induire des lésions pulmonaires induites par un ventilateur (2). A cet effet, des souris C57BL / 6 sont anesthésiés et une trachéotomie est effectuée suivie par l&#39;induction de l&#39;ALI par ventilation mécanique. Les souris sont ventilés dans un cadre à la pression contrôlée avec une pression inspiratoire de pointe de 45 mbar plus de 1 - 3 heures. Comme paramètres de résultats, un oedème pulmonaire (humide à sec ratio), la teneur en fluide broncho-albumine, fluide broncho-pulmonaire et le contenu myéloperoxydase des tissus et des échanges gazeux pulmonaires sont évaluées (2). En utilisant cette technique nous avons pu montrer qu&#39;il induit une inflammation pulmonaire aiguë suffisante et permet de distinguer entre les différents groupes de traitement ou de génotypes (1-3, 5). Par conséquent, cette technique peut être utile pour les chercheurs qui poursuivent des mécanismes moléculaires impliqués dans ALI utilisant une approche génétique chez la souris avec le gène ciblé suppression.

Watch this Scientific Journal Video about Pressure Controlled Ventilation to Induce Acute Lung Injury in Mice at JoVE.com

Pressure Controlled Ventilation to Induce Acute Lung Injury in Mice

Un modèle murin de blessure ventilateur pulmonaire induite est un outil important pour étudier une lésion pulmonaire aiguë<em&gt; In vivo</em&gt;. Ici, nous rapportons une applicables faciles<em&gt; In situ</em&gt; Modèle pour les lésions pulmonaires aiguës à haute pression de ventilation mécanique pour provoquer l&#39;échec aigu du poumon.

La ventilation à pression contrôlée pour induire lésion pulmonaire aiguë chez la souris

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged ...

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18.2K vues.  University of Colorado. Un modèle murin de blessure ventilateur pulmonaire induite est un outil important pour étudier une lésion pulmonaire aiguë In vivo. Ici, nous rapportons une applicables faciles In situ Modèle pour les lésions pulmonaires aiguës à haute pression de ventilation mécanique pour provoquer l'échec aigu du poumon.

Vidéo: Pressure Controlled Ventilation to Induce Acute Lung Injury in Mice

Controlled Cervical Laceration Injury in Mice

Use of genetically modified mice enhances our understanding of molecular mechanisms underlying several neurological disorders such as a spinal cord injury (SCI). Freehand manual control used to produce a laceration model of SCI creates inconsistent injuries often associated with a crush or contusion component and, therefore, a novel technique was developed. Our model of cervical laceration SCI has resolved inherent difficulties with the freehand method by incorporating 1) cervical vertebral stabilization by vertebral facet fixation, 2) enhanced spinal cord exposure, and 3) creation of a reproducible laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm in depth without associated contusion. Compared to the standard methods of creating a SCI laceration such as freehand use of a scalpel or scissors, our method has produced a consistent lesion. This method is useful for studies on axonal regeneration of corticospinal, rubrospinal, and dorsal ascending tracts.

A novel technique to create a reproducible in vivo model of cervical spinal cord laceration injury in the mouse is described. This technique is based on spine stabilization by fixation of the cervical facets and laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm.

Contrôlé blessures de lacération du col chez la souris

Use of genetically modified mice enhances our  understanding of molecular mechanisms underlying several neurological disorders  such as a spinal cord ...

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Médecine

Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often unreported mortality rates that may confound analyses. Bilateral renal pedicle clamping is commonly used to induce IR-AKI, but differences between effective clamp pressures and/or renal responses to ischemia between kidneys often lead to more variable results. In addition, shorter clamp times are known to induce more variable tubular injury, and while mice undergoing bilateral injury with longer clamp times develop more consistent tubular injury, they often die within the first 3 days after injury due to severe renal insufficiency. To improve post-injury survival and obtain more consistent and predictable results, we have developed two models of unilateral ischemia-reperfusion injury followed by contralateral nephrectomy. Both surgeries are performed using a dorsal approach, reducing surgical stress resulting from ventral laparotomy, commonly used for mouse IR-AKI surgeries. For induction of moderate injury BALB/c mice undergo unilateral clamping of the renal pedicle for 26 min and also undergo simultaneous contralateral nephrectomy. Using this approach, 50-60% of mice develop moderate AKI 24 hr after injury but 90-100% of mice survive. To induce more severe AKI, BALB/c mice undergo renal pedicle clamping for 30 min followed by contralateral nephrectomy 8 days after injury. This allows functional assessment of renal recovery after injury with 90-100% survival. Early post-injury tubular damage as well as post injury fibrosis are highly consistent using this model.

We describe models of moderate and severe ischemia-reperfusion-induced kidney injury in which the mice undergo unilateral renal pedicle clamping followed by simultaneous or delayed contralateral nephrectomy, respectively. These models consistently give rise to renal dysfunction and post-injury fibrosis, but injury severity and survival are dependent on mouse background, age and surgical equipment.

Ischémie-reperfusion modèle de lésions rénales aiguës et post blessures fibrose chez la souris

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often ...

Controlled Cortical Impact Model for Traumatic Brain Injury

Every year over a million Americans suffer a traumatic brain injury (TBI). Combined with the incidence of TBIs worldwide, the physical, emotional, social, and economical effects are staggering. Therefore, further research into the effects of TBI and effective treatments is necessary. The controlled cortical impact (CCI) model induces traumatic brain injuries ranging from mild to severe. This method uses a rigid impactor to deliver mechanical energy to an intact dura exposed following a craniectomy. Impact is made under precise parameters at a set velocity to achieve a pre-determined deformation depth. Although other TBI models, such as weight drop and fluid percussion, exist, CCI is more accurate, easier to control, and most importantly, produces traumatic brain injuries similar to those seen in humans. However, no TBI model is currently able to reproduce pathological changes identical to those seen in human patients. The CCI model allows investigation into the short-term and long-term effects of TBI, such as neuronal death, memory deficits, and cerebral edema, as well as potential therapeutic treatments for TBI.

Traumatic brain injuries (TBIs) remain a serious health problem. Using the controlled cortical impact surgery model, research on the effects of TBI and possible treatment methods may be performed.

Contrôlée corticale modèle d&#39;impact pour Traumatic Brain Injury

Every year over a million Americans suffer a traumatic brain injury (TBI).  Combined with the incidence of TBIs worldwide, the physical, emotional, ...

Cardiac Catheterization in Mice to Measure the Pressure Volume Relationship: Investigating the Bowditch Effect

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these strategies is to examine their effects on heart function. There are several techniques to measure in vivo cardiac mechanics (e.g., echocardiography, pressure/volume relations, etc.). Compared to echocardiography, real-time left ventricular (LV) pressure/volume analysis via catheterization is more precise and insightful in assessing LV function. Additionally, LV pressure/volume analysis provides the ability to instantaneously record changes during manipulations of contractility (e.g., &#946;-adrenergic stimulation) and pathological insults (e.g., ischemia/reperfusion injury). In addition to the maximum (+dP/dt) and minimum (-dP/dt) rate of pressure change in the LV, an accurate assessment of LV function via several load-independent indexes (e.g., end systolic pressure volume relationship and preload recruitable stroke work) can be attained. Heart rate has a significant effect on LV contractility such that an increase in the heart rate is the primary mechanism to increase cardiac output (i.e., Bowditch effect). Thus, when comparing hemodynamics between experimental groups, it is necessary to have similar heart rates. Furthermore, a hallmark of many cardiomyopathy models is a decrease in contractile reserve (i.e., decreased Bowditch effect). Consequently, vital information can be obtained by determining the effects of increasing heart rate on contractility. Our and others data has demonstrated that the neuronal nitric oxide synthase (NOS1) knockout mouse has decreased contractility. Here we describe the procedure of measuring LV pressure/volume with increasing heart rates using the NOS1 knockout mouse model.

This article describes the measurement of murine left ventricular function via pressure/volume analysis at different heart rates.

Cathétérisme cardiaque chez la souris pour mesurer le volume de pression Relation: Étudier les effets Bowditch

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these ...

Open Tracheostomy Gastric Acid Aspiration Murine Model of Acute Lung Injury Results in Maximal Acute Nonlethal Lung Injury

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute respiratory distress syndrome (ARDS), characterized by neutrophilic alveolitis, and injury to both alveolar epithelium and vascular endothelium. Clinically, ARDS is defined by acute onset of hypoxemia, bilateral patchy pulmonary infiltrates and non-cardiogenic pulmonary edema. Human studies have provided us with valuable information about the physiological and inflammatory changes in the lung caused by ARDS, which has led to various hypotheses about the underling mechanisms. Unfortunately, difficulties determining the etiology of ARDS, as well as a wide range of pathophysiology have resulted in a lack of critical information that could be useful in developing therapeutic strategies.
Translational animal models are valuable when their pathogenesis and pathophysiology accurately reproduce a concept proven in both in vitro and clinical settings. Although large animal models (e.g., sheep) share characteristics of the anatomy of human trachea-bronchial tree, murine models provide a host of other advantages including: low cost; short reproductive cycle lending itself to greater data acquisition; a well understood immunologic system; and a well characterized genome leading to the availability of a variety of gene deletion and transgenic strains. A robust model of low pH induced ARDS requires a murine ALI that targets mainly the alveolar epithelium, secondarily the vascular endothelium, as well as the small airways leading to the alveoli. Furthermore, a reproducible injury with wide differences between different injurious and non-injurious insults is important.
The murine gastric acid aspiration model presented here using hydrochloric acid employs an open tracheostomy and recreates a pathogenic scenario that reproduces the low pH pneumonitis injury in humans. Additionally, this model can be used to examine interaction of a low pH insult with other pulmonary injurious entities (e.g., food particles, pathogenic bacteria).

This protocol induces acute lung injury in a mouse that has close fidelity to the pathogenesis of acid pneumonitis observed in humans. We generate a maximal acute nonlethal low pH lung injury and account for differences in rodent-human anatomic respiratory structure using an open tracheostomy coupled with circumferential pressure release.

Ouvrir trachéotomie acide gastrique Aspiration modèle murin de lésion pulmonaire aiguë Résultats dans Maximal aiguë non létaux Lung Injury

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute ...

Balloon-based Injury to Induce Myointimal Hyperplasia in the Mouse Abdominal Aorta

The use of animal models is essential for a better understanding of MH, one major cause for arterial stenosis.In this article, we demonstrate a murine balloon denudation model, which is comparable with established vessel injury models in large animals. The aorta denudation model with balloon catheters mimics the clinical setting and leads to comparable pathobiological and physiological changes. Briefly, after performing a horizontal incision in the aorta abdominalis, a balloon catheter will be inserted into the vessel, inflated, and introduced retrogradely. Inflation of the balloon will lead to intima injury and overdistension of the vessel. After removing the catheter, the aortic incision will be closed with single stiches. The model shown in this article is reproducible, easy to perform, and can be established quickly and reliably. It is especially suitable for evaluating expensive experimental therapeutic agents, which can be applied in an economical fashion. By using different knockout-mouse strains, the impact of different genes on MH development can be assessed.

This article demonstrates a murine model to study the development of myointimal hyperplasia (MH) after aortic balloon injury.

Blessure axée sur le ballon pour provoquer l’hyperplasie Myointimal dans l’aorte abdominale de souris

The use of animal models is essential for a better understanding of MH, one major cause for arterial stenosis.In this article, we demonstrate a murine ...

A Closed-chest Model to Induce Transverse Aortic Constriction in Mice

Research on cardiac hypertrophy and heart failure is frequently based on pressure overload mouse models induced by TAC. The standard procedure is to perform a partial thoracotomy to visualize the transverse aortic arch. However, the surgical trauma caused by the thoracotomy in open-chest models changes the respiratory physiology as the ribs are dissected and left unattached after chest closure. To prevent this, we established a minimally invasive, closed chest approach via lateral thoracotomy. Herein we approach the aortic arch via the 2nd intercostal space without entering the chest cavities, leaving the mouse with a less traumatic injury to recover from. We perform this operation using standard laboratory settings for open chest TAC procedures with equal survival rates. Apart from maintaining physiological breathing patterns due to the closed chest approach, the mice seem to benefit by showing rapid recovery, as the less invasive technique appears to facilitate a fast healing process and to reduce immune response after trauma.

Here, we present a protocol of Transverse Aortic constriction (TAC) via a lateral thoracotomy. This technique is a minimally invasive, closed chest surgical procedure aiming to simulate pressure overload and heart failure in mice utilizing standard TAC laboratory settings.

Un modèle à thorax fermé pour induire la Constriction de l’aorte transversale chez les souris

Research on cardiac hypertrophy and heart failure is frequently based on pressure overload mouse models induced by TAC. The standard procedure is to ...

Real-time Pressure-volume Analysis of Acute Myocardial Infarction in Mice

Acute myocardial infarction can lead to acute heart failure and cardiogenic shock. The evaluation of hemodynamics is critical for the evaluation of any potential therapeutic approach directed against acute left ventricular (LV) dysfunction. Current imaging modalities (e.g., echocardiography and magnetic resonance imaging) have several limitations since data on LV pressure cannot directly be measured. LV catheterization in mice undergoing coronary artery occlusion could serve as a novel method for a real-time evaluation of LV function.
At the beginning of the procedure, mice were anesthetized followed by endotracheal intubation. For LV catheterization, the right carotid artery was exposed via middle-neck incision. The catheter was introduced and placed into the LV cavity. Left thoracotomy was conducted and the left main coronary artery (LCA) was ligated. To induce reperfusion, the suture was released after 45 min. Pressure-volume data was recorded at all times.
Ligation of the LCA caused a decrease in LV systolic function as evidenced by a 30% reduction in stroke volume, LV ejection fraction (EF), and cardiac output. Maximum dP/dt as a parameter for LV contractility was also significantly reduced and diastolic function was severely impaired (minimum dP/dt -40%). Reperfusion over a period of 20 min did not lead to a complete recovery of LV function.
Real-time pressure-volume analysis served as a valid procedure for monitoring cardiac function during acute myocardial infarction in mice. Maintaining stable anesthesia and a standardized surgical approach was crucial to ensure valid results. As the early phase of acute myocardial infarction is critical for morbidity and mortality, the delineated method could be beneficial for preclinical evaluation of new strategies for cardioprotection.

Acute myocardial infarction in mice induces acute but incompletely characterized changes in left ventricular (LV) function. LV catheterization in mice undergoing coronary artery occlusion serves as a novel method for a real-time evaluation of LV function.

Analyse en temps réel la pression-volume de l’infarctus aigu du myocarde chez la souris

Acute myocardial infarction can lead to acute heart failure and cardiogenic shock. The evaluation of hemodynamics is critical for the evaluation of any ...

Lung Fixation under Constant Pressure for Evaluation of Emphysema in Mice

Emphysema is a significant feature of chronic obstructive pulmonary disease (COPD). Studies involving an emphysematous mouse model require optimal lung fixation to produce reliable histological specimens of the lung. Due to the nature of the lung&rsquo;s structural composition, which consists largely of air and tissue, there is a risk that it collapses or deflates during the fixation process. Various lung fixation methods exist, each of which has its own advantages and disadvantages. The lung fixation method presented here utilizes constant pressure to enable optimal tissue evaluation for studies using an emphysematous mouse lung model. The main advantage is that it can fix many lungs with the same condition at one time. Lung specimens are obtained from chronic cigarette smoke-exposed mice. Lung fixation is performed using specialized equipment that enables the production of constant pressure. This constant pressure maintains the lung in a reasonably inflated state. Thus, this method generates a histological specimen of the lung that is suitable to evaluate cigarette smoke-induced mild emphysema.

Presented here is a useful protocol for lung fixation that creates a stable condition for histological evaluate of lung specimens from a mouse model of emphysema. The main advantage of this model is that it can fix many lungs with the same constant pressure without lung collapse or deflation.

Fixation pulmonaire sous pression constante pour l'évaluation de l'emphysème chez les souris

Emphysema is a significant feature of chronic obstructive pulmonary disease (COPD). Studies involving an emphysematous mouse model require optimal lung ...

Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. Various approaches have been described, such as the inhalation of aerosolized LPS as well as nasal or intratracheal instillation. The presented protocol describes a detailed step-by-step procedure to induce ALI in mice by direct intratracheal LPS instillation and perform FACS analysis of blood samples, bronchoalveolar lavage (BAL) fluid, and lung tissue. After intraperitoneal sedation, the trachea is exposed and LPS is administered via a 22 G venous catheter. A robust and reproducible inflammatory reaction with leukocyte invasion, upregulation of proinflammatory cytokines, and disruption of the alveolo-capillary barrier is induced within hours to days, depending on the LPS dosage used. Collection of blood samples, BAL fluid, and lung harvesting, as well as the processing for FACS analysis, are described in detail in the protocol. Although the use of the sterile LPS is not suitable to study pharmacologic interventions in infectious diseases, the described approach offers minimal invasiveness, simple handling, and good reproducibility to answer mechanistic immunological questions. Furthermore, dose titration as well as the use of alternative LPS preparations or mouse strains allow modulation of the clinical effects, which can exhibit different degrees of ALI severity or early vs. late onset of disease symptoms.

Presented here is a step-by-step procedure to induce acute lung injury in mice by direct intratracheal lipopolysaccharide instillation and to perform FACS analysis of blood samples, bronchoalveolar lavage fluid, and lung tissue. Minimal invasiveness, simple handling, good reproducibility, and titration of disease severity are advantages of this approach.

Induire une lésion pulmonaire aigue chez la souris par instillation directe du lipopolysaccharide intratrachéal

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. ...