Name: partial heterotopic hindlimb transplantation model in rats
Uploaded: 2021-06-09T14:45:00
Description: Watch this Scientific Journal Video about Partial Heterotopic Hindlimb Transplantation Model in Rats at JoVE.com

Diese Arbeit wurde vom Büro des stellvertretenden Verteidigungsministers für Gesundheitsangelegenheiten durch das Congressionally Directed Medical Research Program unter der Award-Nr. W81XWH-17-1-0680. Meinungen, Interpretationen, Schlussfolgerungen und Empfehlungen sind die der Autoren und werden nicht unbedingt vom Verteidigungsministerium unterstützt.

Alle Tiere erhielten eine humane Pflege gemäß dem NIH-Leitfaden für die Pflege und Verwendung von Labortieren. Das Institutional Animal Care and Use Committee (IACUC-Protokoll 2017N000184) und das Animal Care and Use Review Office (ACURO) genehmigten alle Tierprotokolle. Inzucht männliche Lewis-Ratten (250-400 g) wurden für alle Experimente verwendet.
1. Chirurgie
<ol><li>Betäuben Sie die Lewis-Ratten mit Isofluran-Inhalation. Induzieren Sie die Anästhesie mit 5% Isofluran in der Indu...

<ol>
	<li>Lanteiri, L., 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=Feasibility,+reproducibility,+risks+and+benefits+of+face+transplantation:+a+prospective+study+of+outcomes.">Feasibility, reproducibility, risks and benefits of face transplantation: a prospective study of outcomes.</a> American Journal of Transplantation. 11 (2), 367-378 (2011).</li><li>Park, S. H., Eun, S. C., Kwon, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hand+transplantation:+current+status+and+immunologic+obstacles.">Hand transplantation: current status and immunologic obstacles.</a> Experimental and Clinical Transplantation. 17 (1), 97-104 (2019).</li><li>Cetrulo, C. L., 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=Penis+transplantation:+first+US+experience.">Penis transplantation: first US experience.</a> Annals of Surgery. 267 (5), 983-988 (2018).</li><li>Grajek, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First+complex+allotransplantation+of+neck+organs:+larynx,+trachea,+pharynx,+esophagus,+thyroid,+parathyroid+glands,+and+anterior+cervical+wall:+a+case+report.">First complex allotransplantation of neck organs: larynx, trachea, pharynx, esophagus, thyroid, parathyroid glands, and anterior cervical wall: a case report.</a> Annals of Surgery. 266 (2), 19-24 (2017).</li><li>Pribaz, J. J., Caterson, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+and+limitations+of+conventional+autologous+reconstruction+of+the+head+and+neck.">Evolution and limitations of conventional autologous reconstruction of the head and neck.</a> Journal of Craniofacial Surgery. 24 (1), 99-107 (2013).</li><li>Lipson, R. 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=Vascularized+limb+transplantation+in+the+rat.+I.+Results+with+syngeneic+grafts.">Vascularized limb transplantation in the rat. I. Results with syngeneic grafts.</a> Transplantation. 35 (4), 293-299 (1983).</li><li>Lipson, R. 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=Vascularized+limb+transplantation+in+the+rat.+II.+Results+with+allogeneic+grafts.">Vascularized limb transplantation in the rat. II. Results with allogeneic grafts.</a> Transplantation. 35 (4), 300-304 (1983).</li><li>Adamson, L. 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=A+modified+model+of+hindlimb+osteomyocutaneous+flap+for+the+study+of+tolerance+to+composite+tissue+allografts.">A modified model of hindlimb osteomyocutaneous flap for the study of tolerance to composite tissue allografts.</a> Microsurgery. 27 (7), 630-636 (2007).</li><li>Arav, A., Friedman, O., Natan, Y., Gur, E., Shani, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+hindlimb+cryopreservation+and+transplantation:+a+step+toward+"organ+banking".">Rat hindlimb cryopreservation and transplantation: a step toward "organ banking".</a> American Journal of Transplantation. 17 (11), 2820-2828 (2017).</li><li>Gok, 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=Development+of+an+ex-situ+limb+perfusion+system+for+a+rodent+model.">Development of an ex-situ limb perfusion system for a rodent model.</a> ASAIO Journal. 65 (2), 167-172 (2019).</li><li>Gok, E., Rojas-Pena, A., Bartlett, R. H., Ozer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+skeletal+muscle+metabolomic+changes+associated+with+static+cold+storage.">Rodent skeletal muscle metabolomic changes associated with static cold storage.</a> Transplantation Proceedings. 51 (3), 979-986 (2019).</li><li>Brandacher, G., Grahammer, J., Sucher, R., Lee, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+for+basic+and+translational+research+in+reconstructive+transplantation.">Animal models for basic and translational research in reconstructive transplantation.</a> Birth Defects Research. Part C, Embryo Today. 96 (1), 39-50 (2012).</li><li>Fleissig, Y., 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=Modified+heterotopic+hindlimb+osteomyocutaneous+flap+model+in+the+rat+for+translational+vascularized+composite+allotransplantation+research.">Modified heterotopic hindlimb osteomyocutaneous flap model in the rat for translational vascularized composite allotransplantation research.</a> Journal of Visualized Experiments: JoVE. (146), e59458 (2019).</li><li>Ulusal, A. E., Ulusal, B. G., Hung, L. M., Wei, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterotopic+hindlimb+allotransplantation+in+rats:+an+alternative+model+for+immunological+research+in+composite-tissue+allotransplantation.">Heterotopic hindlimb allotransplantation in rats: an alternative model for immunological research in composite-tissue allotransplantation.</a> Microsurgery. 25 (5), 410-414 (2005).</li><li>Jang, Y., Park, Y. E., Yun, C. W., Kim, D. H., Chung, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+vest-collar+as+a+rodent+collar+to+prevent+licking+and+scratching+during+experiments.">The vest-collar as a rodent collar to prevent licking and scratching during experiments.</a> Lab Anim. 50 (4), 296-304 (2016).</li><li>Kern, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+rodent+orthotopic+forelimb+transplantation+model+that+allows+for+reliable+assessment+of+functional+recovery+resulting+from+nerve+regeneration.">A novel rodent orthotopic forelimb transplantation model that allows for reliable assessment of functional recovery resulting from nerve regeneration.</a> American Journal of Transplantation. 17 (3), 622-634 (2017).</li><li>Perez-Abadia, G., 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=Low-dose+immunosuppression+in+a+rat+hind-limb+transplantation+model.">Low-dose immunosuppression in a rat hind-limb transplantation model.</a> Transplant International. 16 (12), 835-842 (2003).</li><li>Sucher, 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=Orthotopic+hind-limb+transplantation+in+rats.">Orthotopic hind-limb transplantation in rats.</a> Journal of Visualized Experiments. (41), e2022 (2010).</li><li>Fleissig, Y. Y., Beare, J. E., LeBlanc, A. J., Kaufman, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+the+rat+hind+limb+transplant+as+an+experimental+model+of+vascularized+composite+allotransplantation:+Approaches+and+advantages.">Evolution of the rat hind limb transplant as an experimental model of vascularized composite allotransplantation: Approaches and advantages.</a> SAGE Open Medicine. 8, 2050312120968721 (2020).</li><li>Lindboe, C. F., Presthus, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+denervation,+immobilization+and+cachexia+on+fibre+size+in+the+anterior+tibial+muscle+of+the+rat.">Effects of denervation, immobilization and cachexia on fibre size in the anterior tibial muscle of the rat.</a> Acta Neuropathologica. 66 (1), 42-51 (1985).</li><li>Nazzal, J. A., Johnson, T. S., Gordon, C. R., Randolph, M. A., Lee, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterotopic+limb+allotransplantation+model+to+study+skin+rejection+in+the+rat.">Heterotopic limb allotransplantation model to study skin rejection in the rat.</a> Microsurgery. 24 (6), 448-453 (2004).</li></ol>

Orthotopische Gliedmaßentransplantationsmodelle bei Nagetieren wurden in der Literatur15,16,17beschrieben ; Sie erfordern jedoch eine Nervenreparatur, Muskelanhaftung und eine perfekte Osteosynthese des Femurs, was ein sehr schwieriger Schritt sein kann. Diese Modelle sind auch mit einer höheren Morbiditäts- und Mortalitätsrate bei Nagetierenverbunden 14,insbesondere in der kurzfristigen Nachbeobachtu...

In den letzten zwei Jahrzehnten hat sich VCA zu einer revolutionären Behandlung für Patienten entwickelt, die schwere Entstellungen erleiden, einschließlichGesichts-1-und Oberschenkelamputationen2,Penis3und anderer komplexer Gewebedefekte4,5. Die Folgen einer lebenslangen Immunsuppression behindern jedoch immer noch eine breitere Anwendung dieser komplexen rekonstruktiven Operationen. Grundlagenforschung ist entscheidend, um Anti-Ablehnungsstrategien zu verbessern. Die Erhöhung der VCA-Konservierungszeit ist auch wichtig, um die Transplantationslogistik zu verbessern und den Spenderpool zu vergrößern (da VCA-Spender mehr Kriterien erfüllen müssen als feste Organspender, einschließlich Hautton, anatomische Größe, Geschlecht). In diesem Zusammenhang werden Gliedmaßentransplantationen bei Ratten häufig in Studien zur Immunabstoßung von Allotransplantaten6,7, neuartigen Toleranzinduktionsprotokollen8und Konservierungsstudien9,10,11verwendet. Daher sind diese VCA-Modelle ein Schlüsselelement, das es für die translationale VCA-Forschung zu beherrschen gilt.
Osteomyokutane Klappen wurden in der Literatur als zuverlässige Modelle zur Untersuchung von VCA bei Ratten8,12,13,14beschrieben. Obwohl orthotope Ganzgliedmaßentransplantationen eine langfristige Beurteilung der Transplantatfunktion ermöglichen, ist dies ein zeitaufwendiges Verfahren, das mit höheren postoperativen Morbiditäts- und Mortalitätsraten verbunden ist14. Im Gegensatz dazu sind heterotope Gliedmaßentransplantationsmodelle nicht funktionsfähig, ermöglichen aber reproduzierbare Studien zu VCA. Postoperative Ergebnisse können vor Beginn einer VCA-Transplantationsstudie an Ratten zuverlässig antizipiert werden. Diese Studie berichtet über ein partielles heterotopes osteomykutanes Lappentransplantationsmodell bei der Ratte, das häufige mögliche Ergebnisse und Komplikationen beinhaltet, die intraoperativ und postoperativ während einer Nachbeobachtungszeit von drei Wochen auftreten können.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>24 GA angiocatheter</td>
			<td>BD Insyte Autoguard</td>
			<td>381412</td>
			<td></td>
		</tr>
		<tr>
			<td>4-0 suture Black monofilament non absorbable suture</td>
			<td>Ethicon</td>
			<td>1667</td>
			<td>Used to suture the E-collar to the back of the neck</td>
		</tr>
		<tr>
			<td>4-0 suture Coated Vicryl Plus Antibacterial</td>
			<td>Ethicon</td>
			<td>VCP496</td>
			<td></td>
		</tr>
		<tr>
			<td>Adson Tissue Forceps, 11 cm, 1 x 2 Teeth with Tying Platform</td>
			<td>ASSI</td>
			<td>ASSI.ATK26426</td>
			<td></td>
		</tr>
		<tr>
			<td>Bipolar cords</td>
			<td>ASSI</td>
			<td>228000C</td>
			<td></td>
		</tr>
		<tr>
			<td>Black Polyamide Monofilament USP 10-0, 4 mm 3/8c</td>
			<td>AROSurgical</td>
			<td>T04A10N07-13</td>
			<td>Used to perform the microvascular anastomoses</td>
		</tr>
		<tr>
			<td>Buprenorphine HCl</td>
			<td>Pharmaceutical, Inc</td>
			<td>42023-179-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Dilating Forceps</td>
			<td>Fine science tools (FST)</td>
			<td>18131-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting Scissors 15 cm, Round Handle 8 mm diameter, Straight Slender Tapered Blade 7 mm, Lipshultz Pattern</td>
			<td>ASSI</td>
			<td>ASSI.SAS15RVL</td>
			<td></td>
		</tr>
		<tr>
			<td>Double Micro Clamps 5.5 x 1.5 mm</td>
			<td>Fine science tools (FST)</td>
			<td>18040-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Elizabethan collar</td>
			<td>Braintree Scientific</td>
			<td>EC-R1</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps 13.5 cm long, Flat Handle, 9 mm wide Straight Tips 0.1 mm diameter (x2)</td>
			<td>ASSI</td>
			<td>ASSI.JFL31</td>
			<td></td>
		</tr>
		<tr>
			<td>Halsey Micro Needle Holder</td>
			<td>Fine science tools (FST)</td>
			<td>12500-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin Lock Flush Solution, USP, 100 units/mL</td>
			<td>BD PosiFlush</td>
			<td>306424</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Patterson Veterinary</td>
			<td>14043-704-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Jewelers Bipolar Forceps Non Stick 11 cm, straight pointed tip, 0.25 mm tip diameter</td>
			<td>ASSI</td>
			<td>ASSI.BPNS11223</td>
			<td></td>
		</tr>
		<tr>
			<td>Lone Star Elastic Stays</td>
			<td>CooperSurgical</td>
			<td>3314-8G</td>
			<td>Used to retract the inguinal ligament for femoral vessels dissection</td>
		</tr>
		<tr>
			<td>Lone Star Self-Retaining Retractors</td>
			<td>CooperSurgical</td>
			<td>3301G</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-Mosquito Hemostats</td>
			<td>Fine science tools (FST)</td>
			<td>13010-12</td>
			<td>Used to retract the inguinal fat pad distally</td>
		</tr>
		<tr>
			<td>Needle Holder, 15 cm Round Handle, 8 mm diameter, Superfine Curved Jaw 0.2 mm tip diameter, without lock</td>
			<td>ASSI</td>
			<td>ASSI.B1582</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon Suture Black Monolfilament 8-0, 6.5 mm 3/8c</td>
			<td>Ethilon</td>
			<td>2808G</td>
			<td>Used to ligate collateral branches on the femoral vessels</td>
		</tr>
		<tr>
			<td>Offset Bone Nippers</td>
			<td>Fine science tools (FST)</td>
			<td>16101-10</td>
			<td></td>
		</tr>
		<tr>
			<td>S&#38;T Vascular Clamps 5.5 x 1.5 mm</td>
			<td>Fine science tools (FST)</td>
			<td>00398-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Walton scissors</td>
			<td>Fine science tools (FST)</td>
			<td>14077-09</td>
			<td></td>
		</tr>
	</tbody>
</table>

partial heterotopic hindlimb transplantation model in rats

Vaskularisierte kompositative Allotransplantationen (VCA) stellen die fortschrittlichste Rekonstruktionsoption für Patienten ohne autologe Operationsmöglichkeiten nach einem komplexen Gewebedefekt dar. Gesichts- und Handtransplantationen haben das Leben entstellter Patienten verändert und ihnen ein neues ästhetisches und funktionelles soziales Organ gegeben. Trotz vielversprechender Ergebnisse ist VCA aufgrund lebenslanger Immunsuppressionskomorbiditäten und infektiöser Komplikationen immer noch unterdurchschnittlich. Die Ratte ist ein ideales Tiermodell für In-vivo-Studien, die immunologische Wege und Transplantatabstoßungsmechanismen untersuchen. Ratten werden auch häufig in neuartigen Techniken zur Konservierung von Kompositgewebetransplantaten eingesetzt, einschließlich Perfusions- und Kryokonservierungsstudien. Modelle, die für VCA bei Ratten verwendet werden, müssen reproduzierbar, zuverlässig und effizient mit geringer postoperativer Morbidität und Mortalität sein. Heterotope Gliedmaßentransplantationsverfahren erfüllen diese Kriterien und sind einfacher durchzuführen als orthotope Gliedmaßentransplantationen. Die Beherrschung mikrochirurgischer Nagetiermodelle erfordert solide Erfahrung in der Mikrochirurgie und Tierpflege. Hierin wird ein zuverlässiges und reproduzierbares Modell der partiellen heterotopen osteomyokutanen Lappentransplantation bei Ratten, die postoperativen Ergebnisse und die Mittel zur Vorbeugung potenzieller Komplikationen berichtet.

In dieser Einzeloperatorenstudie wurden 30 syngene heterotope partielle Gliedmaßentransplantationen durchgeführt. Erfolg wurde am postoperativen Tag 21 als das Fehlen von VCA-Versagen oder Komplikationen, die Eine Euthanasie erfordern, definiert. Die normale Entwicklung des Transplantats ist in Abbildung 3 dargestellt. Die mittlere Dauer für die partielle Gliedmaßenbeschaffung und den Transplantateintritt beim Empfänger beträgt 35 bzw. 105 Minuten; die mittlere Ischämiezeit betrug 105...

Watch this Scientific Journal Video about Partial Heterotopic Hindlimb Transplantation Model in Rats at JoVE.com

Partial Heterotopic Hindlimb Transplantation Model in Rats

Dieser Artikel stellt ein partielles heterotopes osteomyokutanes Lappentransplantationsprotokoll bei Ratten und seine möglichen Ergebnisse in der mittelfristigen Nachbeobachtung vor.

Partielles heterotopes Hinterbeinein Transplantationsmodell bei Ratten

Vascularized composite allotransplantations (VCA) represent the most advanced reconstruction option for patients without autologous surgical possibilities ...

Wir präsentieren ein zuverlässiges und reproduzierbares operationsmodell der heterotopen Hinterbeinemtransplantation bei Ratten. Diese Technik ist ideal für Kurzzeitstudien zur Konservierung oder Immunabstoßung bei VCA. Die Schritt-für-Schritt-Beschreibung ermöglicht es den Auszubildenden, die Operation schnell zu meistern.Machen Sie zunächst einen umlaufenden Schnitt auf der Haut oberhalb des Knöchels am distalen Drittel des Beins. Skelettieren und kauterisieren Sie die Arteria saphenöses arterie und den terminalen Ast der Arteria poplitea mit einer bipolaren Pinzette. Kauterisieren und schneiden Sie die Muskeln Gastrocnemius, Soleus, Tibialis anterior und Bizeps femoris, bis der Tibiaknochen freigelegt ist.Machen Sie einen 2,5 Zentimeter einschnitt an der rechten Leistenfalte und sezieren Sie das Leistenfettpolster. Verwenden Sie einen Angelhakenretraktor, um das Leistenband zu greifen. Halten Sie das Leistenfettpolster distal mit einer Klemmzange und ziehen Sie das Fettpolster zurück, um die Oberschenkelgefäße freizulegen.Sezieren Sie die Oberschenkelgefäße, individualisieren Sie die Murphy-Äste und ligatieren Sie mit 8/0 Nylonbindern. Heparinisieren Sie die Spenderratte, indem Sie das Heparin mit einer 27,5-Gauge-Nadel in die Penisvene injizieren. Vervollständigen Sie den Hautschnitt um die Hüfte.Verwenden Sie die bipolare Pinzette, um die Muskeln Bizeps femoris und Gluteus superficialis zu kauterisieren, dann kauterisieren und schneiden Sie den Ischiasnerv in mittlerer Femurlänge. Legen Sie den Femur proximal auf Höhe des hinteren Oberschenkelkamms frei und ligaieren Sie die Oberschenkelgefäße mit Nylonbindern auf Höhe des Leistenbandes. Führen Sie eine Arteriotomie an der Oberschenkelarterie direkt unterhalb der Ligatur durch und erweitern Sie sich, um den 24-Gauge-Angiokatheter einzuführen.Kauterisieren und schneiden Sie den verbleibenden Muskel unter dem Pedikel, um die vordere Seite des Femurs freizulegen. Verwenden Sie den Knochenschneider, um die Tibia proximal zu schneiden und den Femur so distal wie möglich zu schneiden. Spülen Sie die partielle Hinterbeine mit zwei Millilitern Heparinsalzlösung, um einen klaren venösen Abfluss zu erhalten.Legen Sie die Hinterbeine bis zum mikrovaskulären Transfer in eine sterile Gaze auf Eis. Sezieren Sie das Leistenfettpolster und lehnen Sie das Leistenfettpolster distal zurück, um die Femurgefäße freizulegen. Verwenden Sie einen Haken, um das Leistenband und eine Klemmzange zurückzuziehen, um das Leistenfettpolster distal zu halten.Sezieren Sie die Oberschenkelgefäße, individualisieren Sie die Murphy-Äste und ligatieren Sie mit Nylonbindern, wie gezeigt. Ligatieren Sie beide Gefäße über den epigastrischen Gefäßen mit Nylonbindern. Setzen Sie die Approximatorklemmen proximal ein, erweitern Sie die Gefäßlinse und spülen Sie sie mit Heparin-Kochsalzlösung ab.Machen Sie einen Schnitt an der linken Flanke oberhalb der Hüfte und erstellen Sie eine subkutane Tasche mit einem subkutanen Tunnel zur Leistenfalte. Legen Sie den proximalen Teil der partiellen Extremität und das Leistenfettpolster durch den subkutanen Tunnel für den mikrovaskulären Transfer. Führen Sie die venösen und arteriellen Anastomosen mit Nylonnähten durch.Entfernen Sie beide Approximatorklemmen und beobachten Sie die Revaskularisierung der Extremität. Führen Sie einen Melktest an beiden Gefäßen durch, um die Durchgängigkeit jeder Anastomose zu beurteilen. Machen Sie einen Längsschnitt auf der medialen Seite der transplantierten Extremität und führen Sie das Transplantat ein.Entfernen Sie die überschüssige Haut des Transplantats und schließen Sie die Wunde mit separaten Nähten und einer laufenden Naht mit resorbierbaren 4/0-Nähten. Nähen Sie die Leistenfettpolster der transplantierten Extremität und des Empfängers mit zwei separaten resorbierbaren Nähten zusammen und schließen Sie die Leistenfalte nach der letzten Untersuchung der mikrovaskulären Anastomose. Die Entwicklung des heterotopen Hinterbeinemodells wurde bis zum Ende der Studie beobachtet.Das Nachwachsen der Haare wurde während der ersten postoperativen Woche beobachtet, und der kutane Rückzug trat nach zwei Wochen auf. Vaskularisierte zusammengesetzte Allotransplantationsversagen kann in der ersten postoperativen Woche aufgrund einer mikrovaskulären Thrombose auftreten. Venenthrombosen waren in 20% der Fälle die Ursache für die frühe Euthanasie, die alle vor dem fünften Tag der Postoperativen auftraten.Die Haut erschien blau und wurde jeden Tag dunkler. Selbstverstümmelung ist ein ernstes Problem bei unsinnigen Transplantaten und tritt oft zwischen dem zweiten und siebten Tag der Postoperativen auf. Wenn sie auf weniger als ein Drittel der Transplantatoberfläche beschränkt sind und nur die Haut betreffen, können chirurgisches Debridement und Naht mit nicht resorbierbaren Nähten mit dem Tierarzt besprochen werden.Die Verhinderung der Selbstverstümmelung beruht auf der Verwendung eines E-Kragens, der bis zum siebten Tag der Postoperation am Hals genäht ist, und der Reinigung von Blut oder Kruste an den chirurgischen Wunden des Tieres. Schwere Autophagie mehrerer Schichten des Transplantats führt zur Euthanasie des Tieres. Dermale Zysten traten nach dem postoperativen Tag 14 auf, manchmal mit einem kutanen nekrotischen Zentrum vor der Fistel.Es ist wichtig sicherzustellen, dass die Platzierung des Transplantats das Tier nicht daran hindert, ambulant zu sein, und dass der Pedikel während des Transplantateinsatzes nicht geknickt oder fest ist. Unser Team führte konservierungsstudien an den beschafften Gliedmaßen vor der Transplantation durch. Die Auswirkungen unterschiedlicher Dauer der statischen Kühllagerung wurden ebenso bewertet wie verschiedene Perfusionsprotokolle für Gliedmaßen.

partial-heterotopic-hindlimb-transplantation-model-in-rats

Research

JoVE Journal

Immunology and Infection

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

Mausmodell der CD40-Aktivierung von B-Zellen

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

Forschung

Immunologie und Infektion

Orthotopic Hind-Limb Transplantation in Rats

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

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

Orthotopen Hind-Limb Transplantation in Ratten

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

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that distinguish acute and chronic inflammation in mouse models. With tissue damage or pathogen invasion, neutrophils are the first line of defense, playing a major role in mediating the acute inflammatory response. As the inflammatory reaction progresses, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages, which mediate chronic inflammation and promote tissue repair by removing tissue debris and producing anti-inflammatory cytokines. Intraperitoneal injection of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils. Luminol specifically reacts with the superoxide generated within the phagosomes of neutrophils since bioluminescence results from a myeloperoxidase (MPO) mediated reaction. Lucigenin (bis-N-methylacridinium nitrate) also reacts with superoxide in order to generate bioluminescence. However, lucigenin bioluminescence is independent of MPO and it solely relies on phagocyte NADPH oxidase (Phox) in macrophages during chronic inflammation. Together, luminol and lucigenin allow non-invasive visualization and longitudinal assessment of different phagocyte populations across both acute and chronic inflammatory phases. Given the important role of inflammation in a variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions.

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.

In-vivo-Imaging-Methode zu unterscheiden, akuten und chronischen Entzündungen

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

Langzeitchronik<em&gt; Pseudomonas aeruginosa</em&gt; Airway-Infektion bei Mäusen

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms requires reliable models. Here, we describe models of tolerance to cardiac allograft in rat, induced by blockade of costimulation signals or by upregulation of immunoregulatory molecules through gene transfer. Each of these models allowed in vivo generation of regulatory cells such as regulatory T cells (Tregs), regulatory B cells (Bregs) or regulatory myeloid cells (RegMCs). In this manuscript, we describe two complementary protocols that have been used to identify and define in vitro and in vivo regulatory cell activity to determine their responsibility in tolerance induction and maintenance. First, an in vitro suppressive assay allowed rapid identification of cells with suppressive capacity on effector immune responses in a dose dependent manner, and can be used for further analysis such as cytokine measurement or cytotoxicity. Second, the adoptive transfer of cells from a tolerant treated recipient to a newly irradiated grafted recipient, highlighted the tolerogenic properties of these cells in controlling graft directed immune responses and/or converting new regulatory cells (termed infectious tolerance). These methods are not restricted to cells with known phenotypic markers and can be extended to any cell population. Furthermore, donor directed allospecificity of regulatory cells (an important goal in the field) can be assessed by using third party donor cells or graft either in vitro or in vivo. Finally, to determine the specific tolerogenic capacity of these regulatory cells, we provide protocols to assess the humoral anti-donor antibody responses and the capacity of the recipient to develop humoral responses against new or former known antigens. The models of tolerance described can be used to further characterize regulatory cells, to identify new biomarkers, and immunoregulatory molecules, and are adaptable to other transplantation models or autoimmune diseases in rodent or human.

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

Here, we present a protocol to induce tolerance in transplantation, and assess in vitro and in vivo the suppressive capacity of distinct cell subsets from the recipient and the immune status of the recipient toward donor or exogenous antigens.

In Vitro und In Vivo Beurteilung der T, B und myeloische Zellen Suppressive Aktivität und humorale Reaktionen von Transplantationspatienten

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms ...

A Recovery Cardiopulmonary Bypass Model Without Transfusion or Inotropic Agents in Rats

Cardiopulmonary bypass (CPB) is indispensable in cardiovascular surgery. Despite the dramatic refinement of CPB technique and devices, multi-organ complications related to prolonged CPB still compromise the outcome of cardiovascular surgeries, and may worsen postoperative morbidity and mortality. Animal models recapitulating the clinical usage of CPB enable the clarification of the pathophysiological processes that occur during CPB, and facilitate pre-clinical studies to develop strategies protecting against these complications. Rat CPB models are advantageous because of their greater cost-effectiveness, convenient experimental processes, abundant testing methods at the genetic or protein levels, and genetic consistency. They can be used for investigating the immune system activation and synthesis of proinflammatory cytokines, compliment activation, and production of oxygen free radicals. The rat models have been refined and have gradually taken the place of large-animal models. Here, we describe a simple CPB model without transfusion and/or inotropic agents in a rat. This recovery model allows the study of the long-term multiple organ sequelae of CPB.

Here, we present a protocol to describe a simple recovery cardiopulmonary bypass model without transfusion or inotropic agents in a rat. This model allows the study of the long-term multiple organ sequelae of cardiopulmonary bypass.

Ein Wiederherstellungsmodell kardiopulmonalen Bypass ohne Transfusion oder inotrope Substanzen bei Ratten

Cardiopulmonary bypass (CPB) is indispensable in cardiovascular surgery. Despite the dramatic refinement of CPB technique and devices, multi-organ ...

An Immunological Model for Heterotopic Heart and Cardiac Muscle Cell Transplantation in Rats

Heterotopic heart transplantation in rats has been a commonly used model for diverse immunological studies for more than 50 years. Several modifications have been reported since the first description in 1964. After 30 years of performing heterotopic heart transplantation in rats, we have developed a simplified surgical approach, which can be easily taught and performed without further surgical training or background.
After dissection of the ascending aorta and the pulmonary artery and ligation of superior and inferior caval and pulmonary veins, the donor heart is harvested and subsequently perfused with ice-cold saline solution supplemented with heparin. After clamping and incising the recipient abdominal vessels, the donor ascending aorta and pulmonary artery are anastomosed to the recipient abdominal aorta and inferior vena cava, respectively, using continuous running sutures.
Depending on different donor-recipient combinations, this model allows analyses of either acute or chronic rejection of allografts. The immunological significance of this model is further enhanced by a novel approach of in-ear injection of vital cardiac muscle cells and subsequent analysis of draining cervical lymphatic tissue.

We describe a model of heterotopic abdominal heart transplantation in rats, implying modifications of current strategies, which lead to a simplified surgical approach. Additionally, we describe a novel rejection model by in-ear injection of vital cardiac muscle cells, allowing further transplant immunological analyses in rats.

Ein immunologisches Modell für heterotopische Herz- und Herzmuskelzelltransplantation bei Ratten

Heterotopic heart transplantation in rats has been a commonly used model for diverse immunological studies for more than 50 years. Several modifications ...

Optimization of the Cuff Technique for Murine Heart Transplantation

Murine cardiac transplantation has been performed for more than 40 years. With advancements in microsurgery, certain new techniques have been used to improve surgical efficiency. In our lab, we have optimized the cuff technique with two major steps. First, we used the inner tube technique to insert a temporary inner tube into the external jugular vein and carotid artery blood vessel to facilitate eversion of the vessel over the cuff. Second, we performed complete heterotopic cardiac transplantation through the collaboration of two experienced surgeons. These modifications effectively reduced the operation time to 25 minutes, with a success rate of 95%. In this report, we describe these procedures in detail and provide a supplemental video. We believe that this report on the improved cuff technique will offer practical guidance for murine heterotopic heart transplantation and will enhance the utility of this mouse model for basic research.

We introduce an inner tube approach to the cuff technique for mouse cervical heterotopic heart transplantation to help evert the vessel over the cuff. We found that cooperation between two experienced surgeons markedly shortens the operation time.

Optimierung der Manschettentechnik für DieMurine Heart Transplantation

Murine cardiac transplantation has been performed for more than 40 years. With advancements in microsurgery, certain new techniques have been used to ...

Bone Marrow Transplantation Procedures in Mice to Study Clonal Hematopoiesis

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor cells (HSPCs). Mutations in driver genes, that confer cellular fitness, can lead to the development of expanding HSPC clones that increasingly give rise to progeny leukocytes harboring the somatic mutation. Because clonal hematopoiesis has been associated with heart disease, stroke, and mortality, the development of experimental systems that model these processes is key to understanding the mechanisms that underly this new risk factor. Bone marrow transplantation procedures involving myeloablative conditioning in mice, such as total-body irradiation (TBI), are commonly employed to study the role of immune cells in cardiovascular diseases. However, simultaneous damage to the bone marrow niche and other sites of interest, such as the heart and brain, is unavoidable with these procedures. Thus, our lab has developed two alternative methods to minimize or avoid possible side effects caused by TBI: 1) bone marrow transplantation with irradiation shielding and 2) adoptive BMT to non-conditioned mice. In shielded organs, the local environment is preserved allowing for the analysis of clonal hematopoiesis while the function of resident immune cells is unperturbed. In contrast, the adoptive BMT to non-conditioned mice has the additional advantage that both the local environments of the organs and the hematopoietic niche are preserved. Here, we compare three different hematopoietic cell reconstitution approaches and discuss their strengths and limitations for studies of clonal hematopoiesis in cardiovascular disease.

We describe three methods of bone marrow transplantation (BMT): BMT with total-body irradiation, BMT with shielded irradiation, and BMT method with no pre-conditioning (adoptive BMT) for the study of clonal hematopoiesis in mouse models.

Knochenmarktransplantationsverfahren bei Mäusen zur Untersuchung der klonalen Hämatopoese

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor ...

Purification and Expansion of Mouse Invariant Natural Killer T Cells for in vitro and in vivo Studies

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or microbial lipid antigens presented by the non-polymorphic MHC class I-related molecule CD1d. Preclinical and clinical studies support a role for iNKT cells in cancer, autoimmunity and infectious diseases. iNKT cells are very conserved throughout species and their investigation has been facilitated by mouse models, including CD1d-deficient or iNKT-deficient mice, and the possibility to unequivocally detect them in mice and men with CD1d tetramers or mAbs specific for the semi-invariant TCR. However, iNKT cells are rare and they need to be expanded to reach manageable numbers for any study. Because the generation of primary mouse iNKT cell line in vitro has proven difficult, we have set up a robust protocol to purify and expand splenic iNKT cells from the iV&#945;14-J&#945;18 transgenic mice (iV&#945;14Tg), in which iNKT cells are 30 times more frequent. We show here that primary splenic iV&#945;14Tg iNKT cells can be enriched through an immunomagnetic separation process, yielding about 95-98% pure iNKT cells. The purified iNKT cells are stimulated by anti-CD3/CD28 beads plus IL-2 and IL-7, resulting in 30-fold expansion by day +14 of the culture with 85-99% purity. The expanded iNKT cells can be easily genetically manipulated, providing an invaluable tool to dissect mechanisms of activation and function in vitro and, more importantly, also upon adoptive transfer in vivo.

We describe a rapid and robust protocol to enrich invariant natural killer T (iNKT) cells from mouse spleen and expand them in vitro to suitable numbers for in vitro and in vivo studies.

Aufreinigung und Expansion von mausinvarianten natürlichen Killer-T-Zellen für in vitro und in vivo Studien

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or ...