Ce travail a été soutenu par un programme de H &amp; M Lee de recherche des résidents chirurgicale Scholars (à JAN), un AO Foundation Start-Up subvention S-12-03M (à LSM) et les Instituts nationaux de la santé subvention R01-AI078910 (à LSM) .

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JAM, BNT, EL, NZ, KPF sont employés de PerkinElmer, qui fabrique des instruments d&#39;imagerie payé, à condition que le Xen29 bioluminescente S. souche aureus, et payé les frais de publication de cette vidéo-article. Les auteurs n&#39;ont rien à révéler.

imagerie multimodalité tels que les techniques d&#39;imagerie qui utilisent l&#39;imagerie optique in vivo en association avec l&#39;imagerie μCT fournit une nouvelle approche technologique qui permet la visualisation 3D, la quantification et le suivi longitudinal des processus biologiques dans un contexte anatomique 1-4. Les protocoles dans la présente étude fournissent des informations détaillées sur la façon in vivo de bioluminescence et l&#39;imagerie de fluorescence peut être combiné avec l&#39;imagerie μCT dans un modèle d&#39;infection orthopédique prothétique d&#39;implant chez la souris pour contrôler la charge bactérienne, une inflammation neutrophile et modifications anatomiques dans l&#39;os de façon non invasive et longitudinalement sur temps. Pris ensemble, les informations obtenues en combinant l&#39;imagerie optique et structurelle représente une avancée technologique majeure, qui peut être particulièrement bien adapté pour étudier les processus biologiques et les conditions pathologiques qui affectent le système musculo-squelettique. Un intérêtconclusion tion qui doit être souligné, c&#39;est que nous avons observé que les signaux fluorescents EGFP-neutrophiles a diminué à des niveaux de fond en 14-21 jours et sont restés à des niveaux de fond pour la durée de l&#39;expérience, malgré la présence de bactéries bioluminescentes. Il est peu probable que l&#39;irradiation aux rayons X impacté survie de neutrophiles comme nous avons observé une cinétique similaire des signaux de neutrophiles chez les souris non irradiées 19. Dans nos précédents travaux impliquant un modèle de S. plaies infectées aureus, l&#39;infiltration de neutrophiles ont consisté en une combinaison de recrutement des neutrophiles de la circulation solide, la survie prolongée des neutrophiles au site d&#39;infection et la domiciliation des cellules progénitrices de KIT + de l&#39;abcès, où elles donnent lieu à mûrir localement neutrophiles 23. Il est probable que des procédés similaires ont contribué à l&#39;infiltration de neutrophiles dans l&#39;implant orthopédique S. modèle d&#39;infection aureus. Bien qu&#39;il ne sait pas pourquoi les signaux de neutrophiles a diminué dans le Orthopmodèle d&#39;infection aedic, il se pourrait que la réponse immunitaire a changé au fil du temps que cette infection a progressé d&#39;un aigu à l&#39;infection chronique et c&#39;est un sujet de recherches futures. Il ya des limites à ce modèle de souris de l&#39;infection articulation prothétique orthopédique et la multimodalité vivo imagerie qui devrait être noté. Tout d&#39;abord, ce modèle de souris est une simplification des procédures réelles et les matériaux utilisés en chirurgie orthopédique chez l&#39;homme 24. Néanmoins, ce modèle fait récapituler l&#39;infection chronique et l&#39;inflammation qui s&#39;ensuit dans l&#39;os et les tissus des articulations que l&#39;on voit dans les infections d&#39;implants orthopédiques droits de 8,9. En outre, pour obtenir les images μCT, des doses relativement faibles d&#39;irradiation aux rayons X ont été utilisées pour minimiser tout effet néfaste sur la santé des animaux au cours de l&#39;infection. Pour une meilleure résolution de l&#39;os, des doses plus élevées de rayonnement de rayons X peuvent être utilisés pour l&#39;imagerie μCT sur un euthanasiésNIMAUX. Cependant, cela éliminerait la possibilité de surveiller de manière non invasive et longitudinalement les modifications de l&#39;os au cours de la durée des expériences. En conclusion, l&#39;imagerie multimodalité impliquant la combinaison de l&#39;ensemble de l&#39;imagerie optique in vivo animaux à l&#39;imagerie anatomique μCT a permis une information plus complète sur l&#39;infection et la réponse inflammatoire. En outre, ces techniques ont permis l&#39;évaluation des conséquences de l&#39;infection et de l&#39;inflammation de l&#39;os et du tissu articulaire. Les travaux futurs pourraient profiter de l&#39;imagerie multimodalité pour évaluer l&#39;efficacité des thérapies antimicrobiennes, les réponses immunitaires, pathogenèse de la maladie et les changements réactifs dans l&#39;os comme nous avons commencé à enquêter 14-18. En outre, l&#39;imagerie multimodale peut évaluer des sondes et des traceurs pour diagnostiquer la présence d&#39;une infection, comme décrit précédemment dans des modèles animaux d&#39;infection de la cuisse, une endocardite, infecter pulmonaireions et les infections biomatériaux 25-28. Enfin, l&#39;utilisation de l&#39;imagerie multimodalité pourrait être étendu au-delà des maladies infectieuses et utilisé dans toutes les disciplines, y compris l&#39;orthopédie, la rhumatologie et l&#39;oncologie, pour enquêter sur d&#39;autres conditions qui affectent le système musculo-squelettique, comme le cancer du squelette, la maladie métastatique, les fractures et l&#39;arthrite 5-7 .

Multimodalité des techniques d&#39;imagerie préclinique qui impliquent la combinaison de l&#39;information optique anatomique et permettent la visualisation et le contrôle des phénomènes biologiques en 3D 4.1. Depuis imagerie μCT permet la visualisation exquis de l&#39;anatomie osseuse, en utilisant l&#39;imagerie μCT en conjonction avec de l&#39;imagerie optique représente une combinaison unique qui pourrait être particulièrement utile pour les processus qui impliquent la biologie osseuse 5-7 de l&#39;enquête. Un exemple serait d&#39;utiliser ces techniques pour étudier les infections d&#39;implants orthopédiques, qui représentent une complication catastrophique en suivant les procédures de chirurgie orthopédique 8,9. Les bactéries forment des biofilms sur les corps étrangers implantés qui favorisent la survie des bactéries en agissant comme une barrière physique qui empêche la détection de cellules du système immunitaire de l&#39;infection et bloque l&#39;accès à des antibiotiques de la bactérie 10,11. L&#39;infection chronique et persistante du tissu articulaire (arthrite septique) uned os (ostéomyélite) induit la résorption osseuse qui entraîne le relâchement de la prothèse et une éventuelle défaillance 8,9. Cette ostéolyse périprothétique résultante est associée à une augmentation de la morbidité et de la mortalité 12,13. Dans notre travail précédent, bioluminescence in vivo et l&#39;imagerie de fluorescence a été utilisé avec des rayons X et l&#39;imagerie micro-tomodensitométrie (μCT) dans un modèle d&#39;infection de prothèse orthopédique commune chez les souris de 14 à 19. Ce modèle consiste à placer un fil de Kirschner titane (K-fil) de telle sorte que l&#39;extrémité élargie de l&#39;implant dans l&#39;articulation du genou à partir de fémurs de souris 14-19 de coupe. Un inoculum de Staphylococcus aureus (souche bioluminescente ou Xen29 Xen36) a ensuite été introduit à la pipette sur la surface de l&#39;implant dans l&#39;articulation du genou avant le site chirurgical est fermé 14 à 19. Imagerie optique in vivo a été utilisé pour détecter et quantifier les signaux bioluminescents, qui correspond à la numbre de bactéries dans le tissu infecté articulations et des os de 14 à 19. En outre, l&#39;imagerie de fluorescence in vivo dans des souris LysEGFP, qui possèdent neutrophiles fluorescents 20, a été utilisé pour quantifier le nombre de neutrophiles qui ont émigré à l&#39;articulation du genou infectées contenant les implants K-fil 14,19. Enfin, les modalités d&#39;imagerie anatomiques, y compris l&#39;imagerie haute résolution des rayons X et l&#39;imagerie μCT, autorisés 2D respective et l&#39;imagerie anatomique 3D de l&#39;os affecté pendant toute la durée de l&#39;infection chronique, qui nous arbitrairement fin généralement entre 2 et 6 semaines post-opératoires 16 , 18. En utilisant ce modèle, l&#39;efficacité de la thérapie antimicrobienne locale et systémique, des réponses immunitaires protectrices et des changements pathologiques dans anatomiques os pourrait être évalué de 14 à 18. Dans ce manuscrit, les protocoles détaillés concernant les modalités d&#39;imagerie optique et μCT dans ce modèle d&#39;infection de prothèse conjointe orthopédique ont été fournis dans deux psystème pour étudier les processus biologiques dans le contexte anatomique de l&#39;os ve. Il s&#39;agit notamment des interventions chirurgicales pour modéliser une infection de l&#39;articulation prothétique orthopédique chez la souris, 2D et 3D en imagerie optique in vivo (pour détecter les signaux bioluminescents bactériennes et les signaux de neutrophiles fluorescentes), l&#39;acquisition μCT d&#39;imagerie et d&#39;analyse et de co-enregistrement des images optiques 3D avec la images μCT.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Xen36 bioluminescent Staphylococcus aureus strain</td>
			<td>PerkinElmer, Hopkinton, MA</td>
			<td></td>
			<td>Bioluminescent Staphylococcus aureus strain derived from ATCC 49525 (Wright), a clinical isolate from a bacteremia patient</td>
		</tr>
		<tr>
			<td>Tryptic soy broth</td>
			<td>BD Biosciences, Franklin Lakes, NJ</td>
			<td>211825</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Soy Agar</td>
			<td>BD Biosciences, Franklin Lakes, NJ</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr>
			<td>LysEGFP knockin mouse strain</td>
			<td></td>
			<td></td>
			<td>Not commercially available. This strain contains a knockin of enhanced green fluorescence protein (EGFP) into the lysozyme M gene</td>
		</tr>
		<tr>
			<td>Betadine</td>
			<td>Purdue Products, Stamford, CT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Kirschner-wire (titanium, 0.8 mm diameter)</td>
			<td>Synthes, West Chester, PA</td>
			<td>492.08</td>
			<td></td>
		</tr>
		<tr>
			<td>Wire Cutter - Duracut T.C.</td>
			<td>H&#38;H Company, Ontario, Canada</td>
			<td>83-7002</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Baxter, Deerfield, IL</td>
			<td>118718</td>
			<td></td>
		</tr>
		<tr>
			<td>Vicryl 5-0 sutures (P-3 Reverse cutting)</td>
			<td>Ethicon, Summerville, NJ. Purchased through VWR International.</td>
			<td>95056-936</td>
			<td></td>
		</tr>
		<tr>
			<td>Sustained-release Buprenorphine (5 ml - 1 mg/ml)</td>
			<td>Zoopharm, Windsor, CO</td>
			<td></td>
			<td>analgesic</td>
		</tr>
		<tr>
			<td><a href="http://www.perkinelmer.com/Catalog/Product/ID/IVISSPE?utm_source=JoVE&amp;utm_medium=display&amp;utm_content=LST-IDT2014-GLO-Q414-JoVE&amp;utm_campaign=LST-IDT2014-GLO">IVIS Spectrum Imaging System</a></td>
			<td>PerkinElmer, Hopkinton, MA</td>
			<td></td>
			<td>optical in vivo imaging system</td>
		</tr>
		<tr>
			<td><a href="http://www.perkinelmer.com/Catalog/Category/ID/Quantum%20FX%20%20microCT?utm_source=JoVE&amp;utm_medium=display&amp;utm_content=LST-IDT2014-GLO-Q414-JoVE&amp;utm_campaign=LST-IDT2014-GLO">Quantum FX in vivo &#956;CT system</a></td>
			<td>PerkinElmer, Hopkinton, MA</td>
			<td></td>
			<td>&#956;CT in vivo imaging system</td>
		</tr>
		<tr>
			<td><a href="http://www.perkinelmer.com/Catalog/Category/ID/Living%20Image%20Software?utm_source=JoVE&amp;utm_medium=display&amp;utm_content=LST-IDT2014-GLO-Q414-JoVE&amp;utm_campaign=LST-IDT2014-GLO">IVIS SpectrumCT Imaging System</a></td>
			<td>PerkinElmer, Hopkinton, MA</td>
			<td></td>
			<td>combined optical and &#956;CT in vivo imaging system</td>
		</tr>
		<tr>
			<td><a href="http://www.perkinelmer.com/Catalog/Product/ID/119240?utm_source=JoVE&amp;utm_medium=display&amp;utm_content=LST-IDT2014-GLO-Q414-JoVE&amp;utm_campaign=LST-IDT2014-GLO">Living Image Software</a></td>
			<td>PerkinElmer, Hopkinton, MA</td>
			<td></td>
			<td>Image analysis software for in vivo optical imaging</td>
		</tr>
	</tbody>
</table>

combined in vivo optical and &#181;ct imaging to monitor infection, inflammation, and bone anatomy in an orthopaedic implant infection in mice

imagerie multimodalité a émergé comme une approche technologique commun utilisé à la fois dans la recherche préclinique et clinique. Des techniques de pointe qui combinent l&#39;imagerie optique et μCT vivo permettent la visualisation de phénomènes biologiques dans un contexte anatomique. Ces modalités d&#39;imagerie peuvent être particulièrement utiles pour étudier les conditions qui ont une incidence os. En particulier, les infections d&#39;implants orthopédiques sont un problème important dans la chirurgie orthopédique clinique. Ces infections sont difficiles à traiter car les biofilms bactériens se forment sur les matériaux implantés chirurgicalement étrangers, conduisant à une inflammation persistante, l&#39;ostéomyélite et l&#39;ostéolyse éventuelle de l&#39;os entourant l&#39;implant, qui aboutit finalement à son descellement et l&#39;échec. Ici, un modèle de souris d&#39;un implant orthopédique prothétique infecté a été utilisé qui a impliqué la mise en place chirurgicale d&#39;un implant Kirschner-fil dans un canal intramédullaire du fémur, de telle façon que l&#39;extrémité de l&#39;implant eXTended dans l&#39;articulation du genou. Dans ce modèle, les souris LysEGFP, une souche de souris qui a neutrophiles EGFP-fluorescentes, ont été employés en association avec une souche de Staphylococcus aureus bioluminescente, qui émet de la lumière naturelle. Les bactéries ont été inoculées dans les articulations des genoux des souris avant la fermeture du site chirurgical. Bioluminescence in vivo et l&#39;imagerie de fluorescence a été utilisée pour quantifier la charge bactérienne et la réponse inflammatoire des neutrophiles, respectivement. En outre, l&#39;imagerie μCT a été effectuée sur des souris de la même manière que la localisation 3D de la bioluminescence et des signaux optiques fluorescents pourrait être co-enregistré avec les images μCT anatomiques. Afin de quantifier les variations de l&#39;os au cours du temps, le volume osseux externe des fémurs distaux ont été mesurés à des instants spécifiques en utilisant un processus de semi-automatisé à base de segmentation contour. Pris dans leur ensemble, la combinaison de vivo bioluminescente / imagerie de fluorescence dans l&#39;imagerie μCT peut être particulièrement utile fou la surveillance non invasive de l&#39;infection, la réponse inflammatoire et des modifications anatomiques dans l&#39;os au cours du temps.

Dans bioluminescente vivo et l&#39;imagerie de fluorescence Dans la présente étude, le protocole est décrit par le modèle publié antérieurement d&#39;une infection de l&#39;articulation prothétique orthopédique chez les souris 14 à 19, qui implique la mise en place chirurgicale d&#39;un implant en titane K-fil qui s&#39;étend à partir d&#39;un canal intramédullaire du fémur dans le joint en espace 14-19. S. aureus souche bioluminescente Xen29 (1 x 10 3 CFU dans 2 ul de PBS) a été introduit à la pipette directement au-dessus de l&#39;implant en titane d&#39;extrémité dans l&#39;articulation du genou avant la fermeture du site chirurgical 16. Pour visualiser et de quantifier le fardeau et l&#39;influx de neutrophiles bactérienne non invasive chez les souris LysEGFP anesthésiés, toute l&#39;imagerie optique animaux in vivo a été réalisée séquentiellement l&#39;image des signaux bioluminescents des bactéries et les signaux EGFP fluorescentes des neutrophiles infiltrent l&#39;aide de l&#39;animal entier optique SIIV spectre dans vivoSystème d&#39;imagerie à trois jours post-opératoires (par exemple, deux jours, 14 et 28). Les signaux bioluminescents Xen29 de souris infectées sont restées au-dessus des signaux de souris infectées simulées pour la durée de l&#39;expérience (Figure 1A, C) 16 fond. Nos travaux antérieurs ont démontré que les signaux in vivo bioluminescentes estimés de près les chiffres de l&#39;ex vivo UFC isolés du / tissu et adhérente osseuse commune aux implants 17,18. En outre, les signaux de fluorescence EGFP ont été plus élevés que les souris sham-infectées à des moments précoces mais approchés des niveaux de fond au cours de l&#39;infection (figure 2B, C) ​​16. Co-registration 3D de signaux optiques in vivo avec des images μCT Pour visualiser les signaux optiques (c.-à-signaux, et des bactéries bioluminescentes fluorescentes EGFP) dans le contexte anatomique de l&#39;articulation du genou post-chirurgicales in 3D, les images optiques générées à l&#39;aide du système d&#39;imagerie IVIS spectre ont été co-enregistrées avec les images μCT générées en utilisant le système d&#39;imagerie Quantum FX μCT. Cette co-enregistrement peut être réalisé parce que la chambre d&#39;imagerie de la souris peut être inséré dans la machine, soit pour faire en sorte que les souris étaient dans la même orientation exacte. Pour vérifier cette précision, les résultats ont été comparés avec une acquisition d&#39;image effectuée à l&#39;aide du spectre-CT in vivo système d&#39;imagerie IVIS qui intègre à la fois des modalités dans un instrument sans nécessiter de déplacement physique de l&#39;animal. Pour mapper les données optiques sur les images μCT en 3D, nous avons utilisé une tomographie optique algorithme de reconstruction diffuse 16. La reconstruction 3D obtenue est représentée (Film 1). En outre, l&#39;imagerie μCT permis la visualisation et la quantification des modifications corrélatives à la qualité et les dimensions de l&#39;os qui a eu lieu au coursl&#39;infection (figure 2) 16. Comme indiqué précédemment, le volume osseux externe du fémur distal sensiblement augmenté au cours du temps (figure 2A) 16. Pour quantifier ces modifications, l&#39;analyse d&#39;image volumétrique 3D a été réalisée sur l&#39;extrémité distale de 25% de la surface du osseux du fémur et de l&#39;évolution du volume de l&#39;os au cours du temps ont été normalisés au volume initial de l&#39;os. Le volume de l&#39;os extérieur sensiblement augmenté chez les souris infectées, comparativement à des souris infectées simulées (figure 2B) 16. L&#39;augmentation du volume de l&#39;os du fémur distal externe est probablement due à des lésions osseuses causées par l&#39;infection des tissus et des os conjointe, qui ont été observés en utilisant μCT imagerie et l&#39;analyse histologique 16. <img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51612/51612fig1highres.jpg" width="500" /> Figure 1: 2D in vivo </strong&gt; bioluminescente et signaux fluorescents. S. aureus Xen29 ou pas de bactéries (non infectées) ont été inoculées dans l&#39;articulation du genou après placement K-fil et souris LysEGFP ont été imagées en utilisant le système d&#39;imagerie IVIS spectre 16 (A). moyenne in vivo des signaux bioluminescents, telle que mesurée par flux total (photons / s) ± sem. (B) moyenne in vivo EGFP signaux fluorescents, telle que mesurée par le nombre total d&#39;efficacité énergétique (photons / sec) / (uW / cm 2) ± sem. (C) représentant en bioluminescence in vivo et des signaux fluorescents recouvert sur ​​une photographie en noir et blanc l&#39;image des souris. La limite de détection de la charge bactérienne en utilisant l&#39;imagerie bioluminescente in vivo est comprise entre 1 x 10 2 et 1 x 10 3 CFU. * P &lt;0,05, † p &lt;0,01, ‡ p &lt;0,001 de souris Xen29 infectés par rapport à des souris de faux-infectés (test t de Student [bilatéral]). S&#39;il vous plaîtnoter qu&#39;il s&#39;agit d&#39;une figure représentative qui inclut les données publiées précédemment générées à l&#39;aide Xen29 et imagés avec le système d&#39;imagerie IVIS Lumina XR 16. <a href="https://www.jove.com/files/ftp_upload/51612/51612fig1highres.jpg" target="_blank">S&#39;il vous plaît cliquer ici pour voir une version plus grande de cette figure.</a> <img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51612/51612fig2highres.jpg" width="500" /> Figure 2: imagerie 3D μCT. S. aureus Xen29 ou pas de bactéries (non infectées) ont été inoculées dans l&#39;articulation du genou après placement K-fil et les souris ont été imagées en utilisant le Quantum FX in vivo du système μCT. (A) représentant les rendus 3D μCT de Xen29 souris infectée par le (panneaux supérieurs) et les souris sham-infectés (panneaux inférieurs). (B) Pourcentage de changement de volume de l&#39;os externe (distale 25% des fémurs) normalisée à la ti initialemoi Point (moyenne ± SEM). * P &lt;0,05, † p &lt;0,01, ‡ p &lt;0,001 de souris Xen29 infectés par rapport à des souris de faux-infectés (test t de Student [bilatéral]). S&#39;il vous plaît noter que ceci est une figure représentative qui inclut les données publiées précédemment générés en utilisant la souche bioluminescente S. aureus Xen29 et imagée avec le Quantum FX in vivo μCT système d&#39;imagerie 16. <a href="https://www.jove.com/files/ftp_upload/51612/51612fig2highres.jpg" target="_blank">S&#39;il vous plaît cliquer ici pour voir une version plus grande de cette figure.</a> <a href="https://www.jove.com/files/ftp_upload/51612/Movie_1.mpg" target="_blank">Film 1</a> . Représentant 3D anatomique de co-enregistrement des signaux bioluminescents Xen29 et les signaux de fluorescence EGFP-neutrophiles en combinaison avec les images μCT. Les images sont mises en rotation sur l&#39;axe vertical.

Watch this Scientific Journal Video about Combined In vivo Optical and ÂµCT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice at JoVE.com

Combined In vivo Optical and  &amp;#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Ensemble d&#39;imagerie optique et μCT dans un modèle murin de l&#39;infection de l&#39;implant orthopédique, en utilisant un bioluminescente souche génétiquement de Staphylococcus aureus, à condition que la capacité de surveiller de manière non invasive et longitudinalement la dynamique de l&#39;infection bactérienne, ainsi que de la réponse inflammatoire correspondant et modifications anatomiques dans l&#39; os.

Combiné<em&gt; In vivo</em&gt; Optique et μCT imagerie pour surveiller l&#39;infection, l&#39;inflammation, et Bone Anatomy dans une infection orthopédique Implant souris

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in ...

combined-vivo-optical-ct-imaging-to-monitor-infection-inflammation

Research

JoVE Journal

Medicine

Combined In vivo Optical and &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

16.9K vues.  David Geffen School of Medicine at University of California, Los Angeles (UCLA).  Ensemble d'imagerie optique et μCT dans un modèle murin de l'infection de l'implant orthopédique, en utilisant un bioluminescente souche génétiquement de Staphylococcus aureus, à condition que la capacité de surveiller de manière non invasive et longitudinalement la dynamique de l'infection bactérienne, ainsi que de la réponse inflammatoire correspondant et modifications anatomiques dans l' os. 

Vidéo: Combined In vivo Optical and  &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Multi-photon imagerie de l&#39;invasion des cellules tumorales dans un modèle de souris orthotopique du carcinome épidermoïde oral

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Recherche

Médecine

Bronchoalveolar Lavage (BAL) for Research; Obtaining Adequate Sample Yield

We describe a research technique for fiberoptic bronchoscopy with bronchoalveolar lavage (BAL) using manual hand held suction in order to remove nonadherent cells and lung lining fluid from the mucosal surface. In research environments, BAL allows sampling of innate (lung macrophage), cellular (B- and T- cells), and humoral (immunoglobulin) responses within the lung.
BAL is internationally accepted for research purposes and since 1999 the technique has been performed in &#62; 1,000 subjects in the UK and Malawi by our group.
Our technique uses gentle hand-held suction of instilled fluid; this is designed to maximize BAL volume returned and apply minimum shear force on ciliated epithelia in order to preserve the structure and function of cells within the BAL fluid and to preserve viability to facilitate the growth of cells in ex vivo culture. The research technique therefore uses a larger volume instillate (typically in the order of 200 ml) and employs manual suction to reduce cell damage.
Patients are given local anesthetic, offered conscious sedation (midazolam), and tolerate the procedure well with minimal side effects. Verbal and written subject information improves tolerance and written informed consent is mandatory. Safety of the subject is paramount. Subjects are carefully selected using clear inclusion and exclusion criteria.
This protocol includes a description of the potential risks, and the steps taken to mitigate them, a list of contraindications, pre- and post-procedure checks, as well as precise bronchoscopy and laboratory techniques.

Bronchoalveolar Lavage BAL for Research; Obtaining Adequate Sample Yield

We describe a research technique for fiberoptic bronchoscopy and bronchoalveolar lavage using low pressure suction. The technique is used to harvest immune cells from the lung bronchoalveolar surfaces. Local anesthetic and mild conscious sedation (midazolam) is used. Subjects tolerate the procedure well and experience minimal side effects.

Lavage broncho-alvéolaire (LBA) pour la recherche; obtention adéquate rendement de l&#39;échantillon

We describe a research technique for fiberoptic bronchoscopy with bronchoalveolar lavage (BAL) using manual hand held suction in order to remove ...

Experimental Human Pneumococcal Carriage

Experimental human pneumococcal carriage (EHPC) is scientifically important because nasopharyngeal carriage of Streptococcus pneumoniae is both the major source of transmission and the prerequisite of invasive disease. A model of carriage will allow accurate determination of the immunological correlates of protection, the immunizing effect of carriage and the effect of host pressure on the pathogen in the nasopharyngeal niche. Further, methods of carriage detection useful in epidemiologic studies, including vaccine studies, can be compared.
Aim
We aim to develop an EHPC platform that is a safe and useful reproducible method that could be used to down-select candidate novel pneumococcal vaccines with prevention of carriage as a surrogate of vaccine induced immunity. It will work towards testing of candidate vaccines and descriptions of the mechanisms underlying EHPC and vaccine protection from carriage1. Current conjugate vaccines against pneumococcus protect children from invasive disease although new vaccines are urgently needed as the current vaccine does not confer optimal protection against non-bacteraemic pneumonia and there has been evidence of serotype replacement with non-vaccine serotypes2-4.
Method
We inoculate with S. pneumoniae suspended in 100 &mu;l of saline. Safety is a major factor in the development of the EHPC model and is achieved through intensive volunteer screening and monitoring. A safety committee consisting of clinicians and scientists that are independent from the study provides objective feedback on a weekly basis.
The bacterial inoculum is standardized and requires that no animal products are inoculated into volunteers (vegetable-based media and saline). The doses required for colonization (104-105) are much lower than those used in animal models (107)5. Detecting pneumococcal carriage is enhanced by a high volume (ideally &gt;10 ml) nasal wash that is relatively mucus free. This protocol will deal with the most important parts of the protocol in turn. These are (a) volunteer selection, (b) pneumococcal inoculum preparation, (c) inoculation, (d) follow-up and (e) carriage detection.
Results
Our current protocol has been safe in over 100 volunteers at a range of doses using two different bacterial serotypes6. A dose ranging study using S. pneumoniae 6B and 23F is currently being conducted to determine the optimal inoculation dose for 50% carriage. A predicted 50% rate of carriage will allow the EHPC model to have high sensitivity for vaccine efficacy with small study numbers.

Experimental human pneumococcal carriage offers a natural model of carriage and a potential model for use in vaccine development. This technique is valuable yet complex and involves clinical risk by introducing a pathogen into a human. We have developed a detailed protocol.

Expérimentale transport pneumocoque humaines

Experimental human pneumococcal carriage (EHPC) is scientifically important because nasopharyngeal carriage of Streptococcus pneumoniae is both the major ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

Infusions de l&#39;artère carotide pour pharmacocinétiques et pharmacodynamiques Analyse des taxanes chez la souris

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of cerebral malaria can be difficult. We present an experimental mouse model of cerebral malaria that shares multiple features of the human disease, including edema and microvascular pathology. Using magnetic resonance imaging (MRI), we can detect and track the blood-brain barrier disruption, edema development, and subsequent brain swelling. We describe multiple MRI techniques that can visualize these pertinent pathological changes. Thus, we show that MRI represents a valuable tool to visualize and track pathological changes, such as edema, brain swelling, and microvascular pathology, in vivo.

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

We describe a mouse model of experimental cerebral malaria and show how inflammatory and microvascular pathology can be tracked in vivo using magnetic resonance imaging.

Le suivi in vivo du développement de l&#39;œdème et de la pathologie microvasculaire dans un modèle de paludisme cérébral expérimental utilisant l&#39;imagerie par résonance magnétique

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of ...

Treatment with Vancomycin Loaded Calcium Sulphate and Autogenous Bone in an Improved Rabbit Model of Bone Infection

Bone infection results from bacterial invasion, which is extremely difficult to treat in clinical, orthopedic, and traumatic surgery. The bone infection may result in sustained inflammation, osteomyelitis, and eventual bone non-union. Establishment of a feasible, reproducible animal model is important to bone infection research and antibiotic treatment. As an in vivo model, the rabbit model is widely used in bone infection research. However, previous studies on rabbit bone infection models showed that the infection status was inconsistent, as the amount of bacteria was variable. This study presents an improved surgical method for inducing bone infection on a rabbit, by blocking the bacteria in the bone marrow. Then, multi-level evaluations can be carried out to verify the modelling method.
In general, debriding necrotic tissue and implantation of vancomycin-loaded calcium sulphate (VCS) are predominant in antibiotic treatment. Although calcium sulphate in VCS benefits osteocyte crawling and new bone growth, massive bone defects occur after debriding. Autogenous bone (AB) is an appealing strategy to overcome bone defects for the treatment of massive bone defects after debriding necrotic bone.
In this study, we used the tail bone as an autogenous bone implanted in the bone defect. Bone repair was measured using micro-computed-tomography (micro-CT) and histological analysis after animal sacrifice. As a result, in the VCS group, bone non-union was consistently obtained. In contrast, the bone defect areas in the VCS-AB group were decreased significantly. The present modeling method described a reproducible, feasible, stable method to prepare a bone infection model. The VCS-AB treatment resulted in lower bone non-union rates after antibiotic treatment. The improved bone infection model and the combination treatment of VCS and autogenous bone could be helpful in studying the underlying mechanisms in bone infection and bone regeneration pertinent to traumatology orthopedic applications.

This study presents an improved rabbit model infected with Staphylococcus aureus by blocking the same amount of bacteria in bone marrow. Vancomycin loaded calcium sulphate and autogenous bone are used for antibiotic and bone repair treatment. The protocol could be helpful for studying bone infection and regeneration.

Traitement par vancomycine chargées de sulfate de Calcium et autogreffe osseuse dans un modèle de lapin amélioré d’Infection osseuse

Bone infection results from bacterial invasion, which is extremely difficult to treat in clinical, orthopedic, and traumatic surgery. The bone infection ...

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. Therefore, adjuvants with both humoral and cellular immuno-stimulatory capabilities that are able to promote cytotoxic T lymphocytes (CTL) responses are urgently needed. Thus, faithful monitoring of adjuvant candidates that induce cross-priming and subsequently enhance CTL generation represents a crucial step in vaccine development. In here we present an application for a method that uses SIINFEKL-specific (OT-I) T cells to monitor the cross-presentation of the model antigen ovalbumin (OVA) in vivo in the presence of different adjuvant candidates. This method represents a rapid test to select adjuvants with the best cross-priming capabilities. The proliferation of CD8+ T cells is the most valuable indication of cross-priming and it is also regarded as a correlate of adjuvant-induced cross-presentation. This feature can be evaluated in different immune organs like lymph nodes and spleen. The extent of the CTL generation can also be monitored, thereby giving insights on the nature of a local (draining lymph node mainly) or a systemic response (distant lymph nodes and/or spleen). This technique further allows multiple modifications for testing drugs that can inhibit specific cross-presentation pathways and also offers the possibility to be used in different strains of conventional and genetically modified mice. In summary, the application that we present here will be useful for vaccine laboratories in industry or academia that develop or modify chemical adjuvants for vaccine research and development.

Rapid In Vivo Assessment of Adjuvant&#039;s Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

We present here an application for a standard immunological technique (CFSE stained OT-I proliferation) intended to rapidly monitor adjuvant-mediated cytotoxic T lymphocyte (CTL) generation in vivo. This fast estimation of CTL capacities is useful for the development of prophylactic vaccines against intracellular pathogens as well as therapeutic cancer vaccines.

Évaluation rapide In Vivo des fonctions de génération de l’Adjuvant T cytotoxique Lymphocytes pour le développement de vaccins

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. ...

The Generation of Closed Femoral Fractures in Mice: A Model to Study Bone Healing

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies that promote efficient bone healing are non-existent and in high demand. Effective and reproducible animal models of fractures healing are needed to understand the complex biological processes associated with bone regeneration. Many animal models of fracture healing have been generated over the years; however, murine fracture models have recently emerged as powerful tools to study bone healing. A variety of open and closed models have been developed, but the closed femoral fracture model stands out as a simple method for generating rapid and reproducible results in a physiologically relevant manner. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice and facilitate a post-fracture stabilization of the femur by inserting an intramedullary steel rod. Although devices such as a nail or a screw offer greater axial and rotational stability, the use of an intramedullary rod provides a sufficient stabilization for consistent healing outcomes without producing new defects in the bone tissue or damaging nearby soft tissue. Radiographic imaging is used to monitor the progression of callus formation, bony union, and subsequent remodeling of the bony callus. Bone healing outcomes are typically associated with the strength of the healed bone and measured with torsional testing. Still, understanding the early cellular and molecular events associated with fracture repair is critical in the study of bone tissue regeneration. The closed femoral fracture model in mice with intramedullary fixation serves as an attractive platform to study bone fracture healing and evaluate therapeutic strategies to accelerate healing.

The murine closed femoral fracture model is a powerful platform to study fracture healing and novel therapeutic strategies to accelerate bone regeneration. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice using an intramedullary steel rod to stabilize the femur.

La génération des Fractures fémorales fermées chez la souris : un modèle pour étudier la guérison osseuse

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies ...

High-Throughput Analysis of Optical Mapping Data Using ElectroMap

Optical mapping is an established technique for high spatio-temporal resolution study of cardiac electrophysiology in multi-cellular preparations. Here we present, in a step-by-step guide, the use of ElectroMap for analysis, quantification, and mapping of high-resolution voltage and calcium datasets acquired by optical mapping. ElectroMap analysis options cover a wide variety of key electrophysiological parameters, and the graphical user interface allows straightforward modification of pre-processing and parameter definitions, making ElectroMap applicable to a wide range of experimental models. We show how built-in pacing frequency detection and signal segmentation allows high-throughput analysis of entire experimental recordings, acute responses, and single beat-to-beat variability. Additionally, ElectroMap incorporates automated multi-beat averaging to improve signal quality of noisy datasets, and here we demonstrate how this feature can help elucidate electrophysiological changes that might otherwise go undetected when using single beat analysis. Custom modules are included within the software for detailed investigation of conduction, single file analysis, and alternans, as demonstrated here. This software platform can be used to enable and accelerate the processing, analysis, and mapping of complex cardiac electrophysiology.

This protocol describes the setup and use of ElectroMap, a MATLAB-based open-source software platform for analysis of cardiac optical mapping data. ElectroMap provides a versatile high-throughput tool for analysis of optical mapping voltage and calcium datasets across a wide range of cardiac experimental models.

Analyse à haut débit des données de cartographie optique à l'aide d'ElectroMap

Optical mapping is an established technique for high spatio-temporal resolution study of cardiac electrophysiology in multi-cellular preparations. Here we ...

Immunofluorescence Labelling of Human and Murine Neutrophil Extracellular Traps in Paraffin-Embedded Tissue

Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMN) due to their lobulated nucleus, are the most abundant type of leukocytes. They mature in the bone marrow and are released into the peripheral blood, where they circulate for about 6&#8722;8 h; however, in tissue, they can survive for days. By diapedesis through the endothelium, they leave the blood stream, enter tissues, and migrate towards the site of an infection following chemotactic gradients.&#160;Neutrophils can combat invading microorganisms by phagocytosis,&#160;degranulation, and generation of neutrophil extracellular traps (NETs). This protocol will help to detect NETs in paraffin-embedded tissue. NETs are the result of a process called NETosis, which leads to the release of nuclear, granular, and cytoplasmic components either from living (vital NETosis) or dying (suicidal NETosis) neutrophils. In vitro, NETs form cloud-like structures, which occupy a space several times larger than that of the cells from which they descended. The backbone of NETs is chromatin, to which a selection of proteins and peptides originating from granules and cytoplasm are bound. Thereby, a high local concentration of toxic compounds is maintained so that NETs can capture and inactivate a variety of pathogens including bacteria, fungi, viruses, and parasites, while diffusion of the highly active NET components leading to damage in neighboring tissue is limited. Nevertheless, in recent years it has become apparent that NETs, if generated in abundance or cleared insufficiently, do have pathological potential ranging from autoimmune diseases to cancer. Thus, detection of NETs in tissue samples may have diagnostic significance, and the detection of NETs in diseased tissue can influence the treatment of patients. Since paraffin-embedded tissue samples are the standard specimen used for pathological analysis, it was sought to establish a protocol for fluorescent staining of NET components in paraffin-embedded tissue using commercially available antibodies.

Neutrophil extracellular traps (NETs) are three-dimensional structures generated by stimulated neutrophil granulocytes. It has become clear in recent years that NETs are involved in a wide variety of diseases. Detection of NETs in tissue may have diagnostic relevance, so standardized protocols for labelling NET components are required.

Immunofluorescence Étiquetage des pièges extracellulaires de l'homme et de la murine Neutrophil dans les tissus paraffins

Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMN) due to their lobulated nucleus, are the most abundant type of leukocytes. They ...