Ce travail a été soutenu par les subventions suivantes à Eva Sevick: NIH R01 CA128919 et les NIH R01 HL092923.

Toutes les études sur les animaux ont été effectuées en conformité avec les normes de l&#39;Université du Texas Health Science Center (Houston, TX), département de médecine comparée et Centre d&#39;imagerie moléculaire, après examen et approbation du protocole par leur soins aux animaux respectifs institutionnel et l&#39;utilisation comité (IACUC) ou Animal Welfare Committee (AWC). 1. Préparation des animaux 24 heures avant à l&#39;imagerie Les étapes ci-dessous doit être fait (le cas échéant) la veille de l&#39;imagerie lymphatique a lieu. <ol><li> Lieu animal dans une boîte à induction et calme avec de l&#39;isoflurane. </li><li> Une fois que l&#39;animal est dans un état d&#39;anesthésie profonde (surveillée avec bout-pincement manœuvre), des animaux sous sédation lieu sur un coussin fessier / peluches et le nez position dans un cône de nez raccordés au gaz isoflurane. </li><li> Pince tous les poils / fourrure (le cas échéant) autour de la zone à imager. </li><li> Appliquer un agent dépilatoire (NAIR) pour la zone rasée et laissez-on la peau pour un maximum de 3 min. </li><li> Essuyez doucement avec tout agent dépilatoire chaude, de la gaze humide ou une serviette en papier. </li><li> Rincer délicatement la peau avec de l&#39;eau tiède et sécher doucement la zone avec de la gaze ou du papier absorbant. </li><li> Permettre aux animaux de récupérer sur un coussin chauffant ou sous une lampe chauffante, et de retourner dans leur cage. </li></ol> 2. Journée de l&#39;imagerie <ol><li> Agent d&#39;imagerie reconstituer avec de l&#39;eau stérile, puis diluer à l&#39;aide stérile, normale (0,85%) pour atteindre une solution saline 645 uM (5 μg/10 pl) pour le GIC ou 200 pM (6,8 μg/10 pl) pour Abd-IRDye800. Conserver les solutions dans l&#39;obscurité les conditions et les utiliser dans les 6 heures suivant la reconstitution. </li><li> Lieu animal dans une boîte à induction et calme avec de l&#39;isoflurane. </li><li> Une fois que l&#39;animal est dans un état d&#39;anesthésie profonde (surveillée avec bout-pincement manœuvre), des animaux sous sédation lieu sur le côté sur un coussin fessier / peluches et le nez position dans un cône de nez raccordés au gaz isoflurane. </li><li> Éteignez les lumières (si la pièce estsombre). Si nécessaire, une lampe de bureau halogène petite peut être utilisée pour une petite quantité de lumière pour voir injections. </li><li> En utilisant une seringue à insuline avec une aiguille 31-gauge, injecter ID 5 pl à 50 pl de l&#39;ICG ou Abd-IRDye800 à la face dorsale de chaque patte arrière et / ou sur le côté gauche et droit de la base de la queue, selon la zone d&#39;intérêt (voir Discussion). Chaque dose injectée peut varier de 0,083 à 1,25 mg / kg (ICG) ou 0.113 à 1,7 mg / kg (Abd-IRDye800). Les volumes d&#39;injection varie avec la souche animale et au site d&#39;injection. Pour les souris athymiques, le volume d&#39;injection peut être de 5 pi (patte arrière) ou 10 pi (base de la queue). Si l&#39;animal n&#39;est pas sous le système d&#39;imagerie pour l&#39;injection (s), placer l&#39;animal dans le cadre du système d&#39;imagerie immédiatement après la injection (s). </li><li> Si aucune absorption de colorant est vu dans les vaisseaux lymphatiques, étape 2.5 sera nécessaire de répéter au besoin selon le protocole animaux. </li><li> Une fois lymphatiques sont vu, le site d&#39;injection avec du ruban électrique noir ou noir papar. </li><li> Acquérir les images lymphatiques pour un maximum de 1 heure à l&#39;aide du logiciel V + + et un petit animal, NIRF système d&#39;imagerie. (Les animaux sont mis sous sédation avec de l&#39;isoflurane et la respiration sont surveillés pendant que les images sont l&#39;acquisition.) Alors que les petits animaux, les imageurs NIRF sont disponibles dans le commerce, nous utilisons une mesure, l&#39;imagerie du petit animal NIRF système constitué d&#39;une diode laser 785-nm (1005-9mm-78503 , Intense, North Brunswick, NJ) équipé d&#39;une lentille asphérique (C24TME-B, Thorlabs, Newton, NJ), un diffuseur (ED1-C20, Thorlabs), et le filtre (LD01-785/10-25, Semrock, Rochester, NY ) pour créer un champ d&#39;excitation uniforme qui éclaire l&#39;animal à un débit de fluence incidente de moins de 1,4 mW par centimètre carré 10. Une multiplication d&#39;électrons à couplage de charges dispositif (EMCCD, PhotonMax512, Princeton Instruments, Trenton, NJ) système de caméra avec deux 830-nm filtres (AND11333, Andover Corp, Salem, NH) et une lentille de 28 mm Nikkor (1992, Nikon, Melville, NY) est utilisé pour capturer des images avec une intégration lymphatiques times de 200 ms, pour l&#39;imagerie dynamique et statique 800 msec pour l&#39;imagerie 5. Voir la figure 1 pour la configuration du système, le tableau pour plus de détails de chaque composant, et la réflexion pour une brève discussion des propriétés imageur clés. </li><li> Permettre aux animaux de récupérer sur un coussin chauffant ou sous une lampe chauffante et de retourner dans leur cage, ou euthanasier. </li><li> Analyser des images en utilisant ImageJ ou un logiciel MATLAB. Voir la Figure 6. </li></ol>

<ol>
	<li>Alitalo, 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+lymphatic+vasculature+in+disease.">The lymphatic vasculature in disease.</a> Nat. Med. 17, 1371-1380 (2011).</li><li>Rasmussen, J. C., Tan, I. C., Marshall, M. V., Fife, C. E., Sevick-Muraca, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphatic+imaging+in+humans+with+near-infrared+fluorescence.">Lymphatic imaging in humans with near-infrared fluorescence.</a> Curr. Opin. Biotechnol. 20, 74-82 (2009).</li><li>Rasmussen, J. C., 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=Human+Lymphatic+Architecture+and+Dynamic+Transport+Imaged+Using+Near-infrared+Fluorescence.">Human Lymphatic Architecture and Dynamic Transport Imaged Using Near-infrared Fluorescence.</a> Transl. Oncol. 3, 362-372 (2010).</li><li>Sevick-Muraca, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translation+of+near-infrared+fluorescence+imaging+technologies:+emerging+clinical+applications.">Translation of near-infrared fluorescence imaging technologies: emerging clinical applications.</a> Annu. Rev. Med. 63, 217-231 (2012).</li><li>Kwon, S., Sevick-Muraca, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+quantitative+imaging+of+lymph+function+in+mice.">Noninvasive quantitative imaging of lymph function in mice.</a> Lymphat. Res. Biol. 5, 219-231 (2007).</li><li>Kwon, S., Sevick-Muraca, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+phenotyping+with+near-infrared+fluorescence+lymphatic+imaging.">Mouse phenotyping with near-infrared fluorescence lymphatic imaging.</a> Biomed Opt Express. 2, 1403-1411 (2011).</li><li>Marshall, M. V., 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=Near-infrared+fluorescence+imaging+in+humans+with+indocyanine+green:+a+review+and+update.">Near-infrared fluorescence imaging in humans with indocyanine green: a review and update.</a> The Open Surgical Oncology Journal. 2, 12-25 (2010).</li><li>Davies-Venn, C. 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=Albumin-Binding+Domain+Conjugate+for+Near-Infrared+Fluorescence+Lymphatic+Imaging.">Albumin-Binding Domain Conjugate for Near-Infrared Fluorescence Lymphatic Imaging.</a> Mol. Imaging Biol. , (2011).</li><li>Sharma, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+imaging+of+lymph+function.">Quantitative imaging of lymph function.</a> Am. J. Physiol. Heart Circ. Physiol. 292, 3109-3118 (2007).</li><li>Kwon, S., Sevick-Muraca, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+lymphatic+imaging+in+tumor-bearing+mice.">Functional lymphatic imaging in tumor-bearing mice.</a> J. Immunol. Methods. 360, 167-172 (2010).</li><li>Karlsen, T. V., McCormack, E., Mujic, M., Tenstad, O., Wiig, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimally+invasive+quantification+of+lymph+flow+in+mice+and+rats+by+imaging+depot+clearance+of+near-infrared+albumin.">Minimally invasive quantification of lymph flow in mice and rats by imaging depot clearance of near-infrared albumin.</a> Am. J. Physiol. Heart Circ. Physiol. 302, 391-401 (2012).</li><li>Zhou, Q., Wood, R., Schwarz, E. M., Wang, Y. J., Xing, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near-infrared+lymphatic+imaging+demonstrates+the+dynamics+of+lymph+flow+and+lymphangiogenesis+during+the+acute+versus+chronic+phases+of+arthritis+in+mice.">Near-infrared lymphatic imaging demonstrates the dynamics of lymph flow and lymphangiogenesis during the acute versus chronic phases of arthritis in mice.</a> Arthritis Rheum. 62, 1881-1889 (2010).</li><li>Adams, K. E., 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=Direct+evidence+of+lymphatic+function+improvement+after+advanced+pneumatic+compression+device+treatment+of+lymphedema.">Direct evidence of lymphatic function improvement after advanced pneumatic compression device treatment of lymphedema.</a> Biomed. Opt. Express. 1, 114-125 (2010).</li><li>Tan, I. C., 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=Assessment+of+lymphatic+contractile+function+after+manual+lymphatic+drainage+using+near-infrared+fluorescence+imaging.">Assessment of lymphatic contractile function after manual lymphatic drainage using near-infrared fluorescence imaging.</a> Arch. Phys. Med. Rehabil. 92, 756-764 (2011).</li><li>Lapinski, P. E., 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=RASA1+maintains+the+lymphatic+vasculature+in+a+quiescent+functional+state+in+mice.">RASA1 maintains the lymphatic vasculature in a quiescent functional state in mice.</a> J. Clin. Invest. 122, 733-747 (2012).</li><li>Maus, E. 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=Near-infrared+fluorescence+imaging+of+lymphatics+in+head+and+neck+lymphedema.">Near-infrared fluorescence imaging of lymphatics in head and neck lymphedema.</a> Head Neck. 34, 448-453 (2012).</li><li>Galanzha, E. I., Tuchin, V. V., Zharov, V. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+small+animal+mesentery+models+for+in+vivo+flow+cytometry,+dynamic+microscopy,+and+drug+screening.">Advances in small animal mesentery models for in vivo flow cytometry, dynamic microscopy, and drug screening.</a> World J. Gastroenterol. 13, 192-218 (2007).</li><li>Schramm, 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+cervical+lymph+node+preparation:+a+novel+approach+to+study+lymphocyte+homing+by+intravital+microscopy.">The cervical lymph node preparation: a novel approach to study lymphocyte homing by intravital microscopy.</a> Inflammation research : official journal of the European Histamine Research Society. 55, 160-167 (2006).</li><li>Hall, M. 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=Imaging+prostate+cancer+lymph+node+metastases+with+a+multimodality+contrast+agent.">Imaging prostate cancer lymph node metastases with a multimodality contrast agent.</a> Prostate. 72, 129-146 (2012).</li><li>Zhu, B., Sevick-Muraca, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimizing+excitation+leakage+and+maximizing+measurement+sensitivity+for+molecular+imaging+with+near-infrared+fluorescence.">Minimizing excitation leakage and maximizing measurement sensitivity for molecular imaging with near-infrared fluorescence.</a> J. Innovat. Opt. Health Sci. 4, 301-307 (2011).</li></ol>

Les auteurs n&#39;ont rien à révéler, mais certains auteurs sont inscrites à un brevet.

Nous utilisons une coutume, petit animal NIRF système d&#39;imagerie pour capturer des images de vaisseaux lymphatiques marquées chez la souris. Pour construire films de mouvement lymphe, plus de 300 images sont recueillies. Pour l&#39;analyse fonctionnelle des vaisseaux lymphatiques de films, deux ou plusieurs ROIs sont manuellement tirée le long d&#39;un vaisseau lymphatique. Les dimensions de la ROI sont maintenues constantes pour chaque navire, et sont à peu près au diamètre de la cuve. Bien que la résolution...

<table border="1"><tbody><tr><td> Solutions, réactifs et équipement </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> Le vert d&#39;indocyanine (ICG) </td><td> Patheon Italia SPA </td><td> NDC 25431-424-02 </td><td> Reconstituer à 645 uM (5 μg/10 pl) </td></tr><tr><td> Cyclique Domaine liaison à l&#39;albumine (Abd) </td><td> Bachem </td><td> Coutume </td><td> Reconstituer à 200 uM (6,8 μg/10 pl) </td></tr><tr><td> IRDye800 </td><td> Li-COR </td><td> IRDye 800CW </td><td> Reconstituer selon les instructions du fabricant; conjuguer avec Abd à des concentrations equilmolar </td></tr><tr><td> De l&#39;eau stérile </td><td> Hospira, Inc, Lake Forest, IL </td><td> NDC 0409-4887-10 </td><td></td></tr><tr><td> NAIR </td><td> Church &amp; Dwight Co.,Inc </td><td> Magasins locaux </td><td> <a href="http://www.nairlikeneverbefore.com" target="_blank">www.nairlikeneverbefore.com</a> </td></tr><tr><td> Système d&#39;imagerie (composants ci-dessous) </td><td> Center for Molecular Imaging </td><td> N / A </td><td> Construit sur mesure dans nos laboratoires. </td></tr><tr><td> Electron-multiplication charge-coupled device (EMCCD) caméra </td><td> Princeton Instruments, Trenton, NJ </td><td> Photon max 512 </td><td></td></tr><tr><td> Nikon objectif de la caméra </td><td> Nikon Inc, Melville, NY </td><td> Modèle n ° 1992, Nikkor 28mm </td><td></td></tr><tr><td> Filtre optique </td><td> Andover Corp, Salem, NH </td><td> ANDV11333 </td><td> Deux filtres passe-bande 830.0/10.0 nm sont utilisés devant l&#39;objectif </td></tr><tr><td> Diode laser 785-nm </td><td> Intense Ltd, North Brunswick, NJ </td><td> 1005-9MM-78503 </td><td> 500 mW de sortie optique </td></tr&gt; <tr><td> Optique de collimation </td><td> Thorlabs, Newton, NJ </td><td> C240TME-B </td><td> Sortie du laser collimate avant filtre de nettoyage </td></tr><tr><td> Nettoyage du filtre </td><td> Semrock, Inc, Rochester, NY </td><td> LD01-785/10-25 </td><td> Supprime émission laser dans la bande de fluorescence </td></tr><tr><td> Diffuseur optique </td><td> Thorlabs, Newton, NJ </td><td> ED1-C20 </td><td> Diffuse le laser au-dessus de l&#39;animal </td></tr><tr><td> V + + </td><td> Optique numérique, Browns Bay, Auckland, Nouvelle-Zélande </td><td> Version 5.0 </td><td> Logiciel utilisé pour contrôler système de caméra et enregistrer des images sur l&#39;ordinateur. <a href="http://digitaloptics.net/" target="_blank">http://digitaloptics.net/</a> </td></tr><tr><td></td><td></td><td></td><td> Logiciel d&#39;analyse ou l&#39;autre des logiciels suivants peuvent être utilisés pour l&#39;analyse d&#39;image </td></tr><tr><td> ImageJ </td><td> National Institutetuts de la santé, Bethesda, MD </td><td> Version la plus récente disponible </td><td> Freeware disponibles à <a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/</a> </td></tr><tr><td> MATLAB </td><td> The MathWorks, Natick, MA </td><td> Version 2008a ou tard </td><td> <a href="http://www.mathworks.com/" target="_blank">http://www.mathworks.com/</a> </td></tr></tbody></table>

non-invasive optical imaging of the lymphatic vasculature of a mouse

Le système vasculaire lymphatique est une composante importante du système circulatoire qui maintient l&#39;homéostasie fluide, assure la surveillance immunitaire, et l&#39;absorption de graisse sert d&#39;intermédiaire dans l&#39;intestin. Pourtant, malgré sa fonction critique, il ya relativement peu de compréhension de la façon dont le système lymphatique s&#39;adapte pour répondre à ces fonctions en matière de santé et de la maladie 1. Récemment, nous avons démontré la capacité de l&#39;architecture dynamiquement l&#39;image lymphatique et de la lymphe &quot;pompage&quot; action sujets humains normaux ainsi que chez les personnes présentant un dysfonctionnement lymphatique en utilisant l&#39;administration trace d&#39;un proche infra-rouge fluorescent (NIRF) colorant et une coutume, Gen III- Système d&#39;imagerie intensifié 2-4. NIRF imagerie ont montré des changements dramatiques dans l&#39;architecture et la fonction lymphatique avec la maladie humaine. Il reste à savoir comment ces changements se produisent et de nouveaux modèles animaux sont développées pour élucider leur base génétique et moléculaire. Dans ce protocole, nous présentons lymphatique NIRF, sanimaux centre d&#39;imagerie utilisant 5,6 vert d&#39;indocyanine (ICG), un colorant qui a été utilisé pendant 50 ans chez l&#39;homme 7, et un NIRF marqué par un colorant cyclique domaine liaison à l&#39;albumine (Abd-IRDye800) peptide qui se lie préférentiellement la souris et l&#39;albumine humaine 8 . Environ 5,5 fois plus brillante que l&#39;ICG, Abd-IRDye800 a un profil similaire lymphatique jeu et peut être injecté dans les petites doses de l&#39;ICG pour atteindre signaux NIRF suffisantes pour l&#39;imagerie 8. Parce que les deux se lient Abd-IRDye800 et ICG à l&#39;albumine dans l&#39;espace interstitiel 8, ils peuvent représenter à la fois le transport des protéines actif dans et à l&#39;intérieur des vaisseaux lymphatiques. Intradermique (ID) injections (ul 5-50) de l&#39;ICG (645 uM) ou Abd-IRDye800 (200 uM) dans une solution saline sont administrés à la face dorsale de chaque patte arrière et / ou sur le côté gauche et droit de la base de la la queue d&#39;une souris anesthésiée isoflurane. La concentration du colorant résultant de l&#39;animal est 83-1,250 mg / kg pour le GIC ou 113-1,700 mg / kg pourAbd-IRDye800. Immédiatement après les injections, imagerie fonctionnelle lymphatique est réalisée pendant 1 h en utilisant un personnalisé, petit animal NIRF système d&#39;imagerie. Résolution spatiale animal entier peut représenter vaisseaux lymphatiques fluorescentes de 100 microns ou moins, et les images des structures jusqu&#39;à 3 cm de profondeur peut être acquis 9. Les images sont acquises à l&#39;aide du logiciel V + + et analysées en utilisant le logiciel MATLAB ou ImageJ. Pendant l&#39;analyse, régions consécutives d&#39;intérêt (ROI) englobant le diamètre du vaisseau entier sont attirés le long d&#39;un vaisseau lymphatique donnée. Les dimensions de chaque ROI sont maintenus constants pour un navire donné et l&#39;intensité NIRF est mesurée pour chaque ROI d&#39;évaluer quantitativement «paquets» de la lymphe se déplaçant à travers les vaisseaux.

Exemple d&#39;imagerie lymphatique NIRF chez la souris Lorsque ICG ou Abd-IRDye800 est injecté ID à la base de la queue d&#39;une souris normale, la vascularisation lymphatique entre le site d&#39;injection à la base de la queue et du ganglion lymphatique inguinal (LN) doit être immédiatement visualisées. Peu de temps après l&#39;injection (quelques secondes à quelques minutes), le vaisseau lymphatique inguinal entre la LN LN et le axillaire doit être visualisé comme le montre la <str...

Watch this Scientific Journal Video about Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse at JoVE.com

Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

Les techniques d&#39;imagerie développées récemment en utilisant la fluorescence dans le proche infrarouge (NIRF) peut aider à élucider le rôle du système lymphatique joue dans les métastases du cancer, la réponse immunitaire, la cicatrisation des plaies, et d&#39;autres maladies associées lymphatique.

Non invasive d&#39;imagerie optique du système vasculaire lymphatique d&#39;une souris

The lymphatic  vascular system is an important component of the circulatory system that  maintains fluid homeostasis, provides immune surveillance, and ...

non-invasive-optical-imaging-of-the-lymphatic-vasculature-of-a-mouse

Research

JoVE Journal

Immunology and Infection

16.0K vues.  University of Texas Health Science Center-Houston. Les techniques d'imagerie développées récemment en utilisant la fluorescence dans le proche infrarouge (NIRF) peut aider à élucider le rôle du système lymphatique joue dans les métastases du cancer, la réponse immunitaire, la cicatrisation des plaies, et d'autres maladies associées lymphatique.

Vidéo: Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

L&#39;imagerie non invasive des Homing leucocytes et des migrations In vivo</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

Recherche

Immunologie et infection

Preparation of Mouse Pituitary Immunogen for the Induction of Experimental Autoimmune Hypophysitis

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1. It has traditionally been considered a rare disease but reporting has increased markedly in recent years. Hypophysitis, in fact, develops not uncommonly as a "side effect" in cancer patients treated with antibodies that block inhibitory receptors expressed on T lymphocytes, such as CTLA-42 and PD-1 receptors. Autoimmune hypophysitis can be induced experimentally by injecting mice with pituitary proteins mixed with an adjuvant3. In this video article we demonstrate how to extract proteins from mouse pituitary glands and how to prepare them in a form suitable for inducing autoimmune hypophysitis in SJL mice.

Autoimmune hypophysitis can be reproduced in mice by injecting an extract of mouse pituitary proteins.

Préparation de la souris Immunogène hypophysaires pour l&#39;induction de hypophysite auto-immune expérimentale

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1.  It has traditionally been considered a ...

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. Historically, intercellular contact events such as phagocytosis3 have been imaged by mixing two cell types, and then continuously scanning the field-of-view to find serendipitous intercellular contacts at the appropriate stage of interaction. The stochastic nature of these events renders this process tedious, and it is difficult to observe early or fleeting events in cell-cell contact by this approach. This method requires finding cell pairs that are on the verge of contact, and observing them until they consummate their contact, or do not. To address these limitations, we use optical trapping as a non-invasive, non-destructive, but fast and effective method to position cells in culture.
Optical traps, or optical tweezers, are increasingly utilized in biological research to capture and physically manipulate cells and other micron-sized particles in three dimensions4. Radiation pressure was first observed and applied to optical tweezer systems in 19705, 6, and was first used to control biological specimens in 19877. Since then, optical tweezers have matured into a technology to probe a variety of biological phenomena8-13.
We describe a method14 that advances live cell imaging by integrating an optical trap with spinning disk confocal microscopy with temperature and humidity control to provide exquisite spatial and temporal control of pathogenic organisms in a physiological environment to facilitate interactions with host cells, as determined by the operator. Live, pathogenic organisms like Candida albicans and Aspergillus fumigatus, which can cause potentially lethal, invasive infections in immunocompromised individuals15, 16 (e.g. AIDS, chemotherapy, and organ transplantation patients), were optically trapped using non-destructive laser intensities and moved adjacent to macrophages, which can phagocytose the pathogen. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability in immunology, primary T-cells were also trapped and manipulated to form synapses with anti-CD3 coated microspheres in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine spatial control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.

A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells.

L&#39;utilisation d&#39;un piège optique pour l&#39;étude des interactions hôtes pathogènes pour l&#39;imagerie cellulaire dynamique en direct

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and ...

A Method for Labeling Vasculature in Embryonic Mice

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in the thymus proper vasculature formation and patterning is essential for thymocyte entry into the organ and mature T-cell exit to the periphery. The spatial arrangement of blood vessels in the thymus is dependent upon signals from the local microenvironment, namely thymic epithelial cells (TEC). Several recent reports suggest that disruption of these signals results in thymus blood vessel defects 1,2. Previous studies have described techniques used to label the neonatal and adult thymus vasculature 1,2. We demonstrate here a technique for labeling blood vessels in the embryonic thymus. This method combines the use of FITC-dextran or Griffonia (Bandeiraea) Simplicifolia Lectin I (GSL 1 - isolectin B4) facial vein injections and CD31 antibody staining to identify thymus vascular structures and PDGFR-&beta; to label thymic perivascular mesenchyme 3-5. The option of using cryosections or vibratome sections is also provided. This protocol can be used to identify thymus vascular defects, which is critical for defining the roles of TEC-derived molecules in thymus blood vessel formation. As the method labels the entire vasculature, it can also be used to analyze the vascular networks in multiple organs and tissues throughout the embryo including skin and heart 6-10.

This article describes a method for labeling embryonic skin and thymus blood vessels.

Une méthode pour l&#39;étiquetage dans les vaisseaux sanguins souris embryonnaires

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in ...

Intravital Imaging of the Mouse Popliteal Lymph Node

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign antigens from peripheral tissues to prime the adaptive immune response. Juxtaposed between innate and adaptive immune responses, the LN is an ideal site to study immune cell interactions1,2. Lymphocytes (T cells, B cells and NK cells), dendritic cells (DCs), and macrophages comprise the bulk of bone marrow-derived cellular elements of the LN. These cells are strategically positioned in the LN to allow efficient surveillance of self antigens and potential foreign antigens3-5. The process by which lymphocytes successfully encounter cognate antigens is a subject of intense investigation in recent years, and involves an integration of molecular contacts including antigen receptors, adhesion molecules, chemokines, and stromal structures such as the fibro-reticular network2,6-12. 
Prior to the development of high-resolution real-time fluorescent in vivo imaging, investigators relied on static imaging, which only offers answers regarding morphology, position, and architecture. While these questions are fundamental in our understanding of immune cell behavior, the limitations intrinsic with this technique does not permit analysis to decipher lymphocyte trafficking and environmental clues that affect dynamic cell behavior. Recently, the development of intravital two-photon laser scanning microscopy (2P-LSM) has allowed investigators to view the dynamic movements and interactions of individual cells within live LNs in situ12-16. In particular, we and others have applied this technique to image cellular behavior and interactions within the popliteal LN, where its compact, dense nature offers the advantage of multiplex data acquisition over a large tissue area with diverse tissue sub-structures11,17-18. It is important to note that this technique offers added benefits over explanted tissue imaging techniques, which require disruption of blood, lymph flow, and ultimately the cellular dynamics of the system. Additionally, explanted tissues have a very limited window of time in which the tissue remains viable for imaging after explant. With proper hydration and monitoring of the animal's environmental conditions, the imaging time can be significantly extended with this intravital technique. Here, we present a detailed method of preparing mouse popliteal LN for the purpose of performing intravital imaging.

Recent advances in 2-photon microscopy have enabled real-time in situ imaging of live tissues in animal models, thereby enhancing our ability to investigate cellular behavior in both physiologic and pathologic conditions. Here, we outline the preparations required to perform intravital imaging of the mouse popliteal lymph node.

Imagerie intravitale du ganglion lymphatique poplité souris

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign ...

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 to 40% attributable mortality6. Systemic candidiasis is normally controlled by innate immunity, and individuals with genetic defects in innate immune cell components such as phagocyte NADPH oxidase are more susceptible to candidemia7-9. Very little is known about the dynamics of C. albicans interaction with innate immune cells in vivo. Extensive in vitro studies have established that outside of the host C. albicans germinates inside of macrophages, and is quickly destroyed by neutrophils10-14. In vitro studies, though useful, cannot recapitulate the complex in vivo environment, which includes time-dependent dynamics of cytokine levels, extracellular matrix attachments, and intercellular contacts10, 15-18. To probe the contribution of these factors in host-pathogen interaction, it is critical to find a model organism to visualize these aspects of infection non-invasively in a live intact host. 
The zebrafish larva offers a unique and versatile vertebrate host for the study of infection. For the first 30 days of development zebrafish larvae have only innate immune defenses2, 19-21, simplifying the study of diseases such as disseminated candidiasis that are highly dependent on innate immunity. The small size and transparency of zebrafish larvae enable imaging of infection dynamics at the cellular level for both host and pathogen. Transgenic larvae with fluorescing innate immune cells can be used to identify specific cells types involved in infection22-24. Modified anti-sense oligonucleotides (Morpholinos) can be used to knock down various immune components such as phagocyte NADPH oxidase and study the changes in response to fungal infection5. In addition to the ethical and practical advantages of using a small lower vertebrate, the zebrafish larvae offers the unique possibility to image the pitched battle between pathogen and host both intravitally and in color. 
The zebrafish has been used to model infection for a number of human pathogenic bacteria, and has been instrumental in major advances in our understanding of mycobacterial infection3, 25. However, only recently have much larger pathogens such as fungi been used to infect larva5, 23, 26, and to date there has not been a detailed visual description of the infection methodology. Here we present our techniques for hindbrain ventricle microinjection of prim25 zebrafish, including our modifications to previous protocols. Our findings using the larval zebrafish model for fungal infection diverge from in vitro studies and reinforce the need to examine the host-pathogen interaction in the complex environment of the host rather than the simplified system of the Petri dish5.

The rapid development, small size and transparency of zebrafish are tremendous advantages for the study of innate immune control of infection1-4. Here we demonstrate techniques for infecting zebrafish larvae using the fungal pathogen Candida albicans by microinjection, methodology recently used to implicate phagocyte NADPH oxidase activity in control of fungal dimorphism5.

L&#39;imagerie non invasive de la candidose disséminée chez les larves poisson zèbre

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 ...

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal features of the natural infection. This protocol describes procedures for the establishment and evaluation of systemic infection due to neuropathogenic Escherichia coli K1 in the neonatal rat. Colonization of the gastrointestinal tract leads to dissemination of the pathogen along the gut-lymph-blood-brain course of infection and the model displays strong age dependency. A strain of E. coli O18:K1 with enhanced virulence for the neonatal rat produces exceptionally high rates of colonization, translocation to the blood compartment and invasion of the meninges following transit through the choroid plexus. As in the human host, penetration of the central nervous system is accompanied by local inflammation and an invariably lethal outcome. The model is of proven utility for studies of the mechanism of pathogenesis, for evaluation of therapeutic interventions and for assessment of bacterial virulence.

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Here, a procedure is described for the establishment of systemic infection in the neonatal rat with cultures of Escherichia coli K1. This non-invasive procedure permits colonization of the gastrointestinal tract, translocation of the pathogen to the systemic circulation, and invasion of the central nervous system at the choroid plexus.

Modèle non-invasive de neuropathogènes<em&gt; Escherichia coli</em&gt; Maladies infectieuses chez le rat nouveau-né

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

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 ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

Imagerie non invasive de la réponse immunitaire innée dans un modèle de poisson zèbre larves de<em&gt; Streptococcus iniae</em&gt; Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under &#39;blind&#39; and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

Une méthode non-invasive et technique non-intensif pour Induction et phénotypage of Experimental pneumonie bactérienne chez les souris

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...