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

Este trabajo fue apoyado por la Oficina del Subsecretario de Defensa para Asuntos de Salud a través del Programa de Investigación Médica Dirigida por el Congreso bajo el Premio No. W81XWH-17-1-0680. Las opiniones, interpretaciones, conclusiones y recomendaciones son las de los autores y no están necesariamente respaldadas por el Departamento de Defensa.

Todos los animales recibieron atención humana de acuerdo con la Guía de los NIH para el Cuidado y Uso de Animales de Laboratorio. El Comité Institucional de Cuidado y Uso de Animales (protocolo IACUC 2017N000184) y la Oficina de Revisión de Cuidado y Uso de Animales (ACURO) aprobaron todos los protocolos de animales. Se utilizaron ratas Lewis macho endogámicas (250-400 g) para todos los experimentos.
1. Cirugía
<ol><li>Anestesiar a las ratas Lewis usando inhalación de isoflurano. 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>

En la literatura se han descrito modelos de trasplante ortotópico de extremidades en roedores15,16,17; sin embargo, requieren una reparación nerviosa, reinserción muscular y una osteosíntesis perfecta del fémur, lo que puede ser un paso muy difícil. Estos modelos también se asocian con una mayor tasa de morbilidad y mortalidad en roedores14,especialmente en el seguimiento a corto plazo ya que la re...

En las últimas dos décadas, VCA ha evolucionado como un tratamiento revolucionario para pacientes que sufren desfiguración severa, incluyendo cara1,amputaciones de miembros superiores2,pene3y otros defectos tisulares complejos4,5. Sin embargo, las consecuencias de la inmunosupresión de por vida aún dificultan una aplicación más amplia de estas complejas cirugías reconstructivas. La investigación básica es crucial para mejorar las estrategias contra el rechazo. Aumentar el tiempo de preservación de VCA también es esencial para mejorar la logística de trasplante y aumentar el grupo de donantes (ya que los donantes de VCA deben cumplir con más criterios que los donantes de órganos sólidos, incluido el tono de piel, el tamaño anatómico y el género). En este contexto, los trasplantes de extremidades de rata son ampliamente utilizados en estudios sobre el rechazo inmune de los aloinjertos6,7,nuevos protocolos de inducción de tolerancia8y estudios de preservación9,10,11. Por lo tanto, estos modelos de VCA son un elemento clave para dominar la investigación traslacional de VCA.
Los colgajos osteomiocutáneos han sido descritos en la literatura como modelos fiables para estudiar el ACV enratas 8,12,13,14. Aunque los trasplantes ortotópicos de extremidades enteras permiten la evaluación a largo plazo de la función del injerto, es un procedimiento que consume mucho tiempo asociado con mayores tasas de morbilidad y mortalidad postoperatoria14. Por el contrario, los modelos heterotópicos de trasplante de extremidades no son funcionales, pero permiten estudios reproducibles sobre el ACV. Los resultados postoperatorios se pueden anticipar de manera confiable antes del inicio de un estudio de trasplante de ACV en rata. Este estudio informa un modelo de trasplante de colgajo osteomiocutáneo heterotópico parcial en la rata que incluye posibles resultados frecuentes y complicaciones que pueden surgir intraoperatoria y postoperatoriamente durante un período de seguimiento de tres semanas.

<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

Los alotrasplantes compuestos vascularizados (ACV) representan la opción de reconstrucción más avanzada para pacientes sin posibilidades quirúrgicas autólogas después de un defecto tisular complejo. Los trasplantes de cara y mano han cambiado la vida de los pacientes, dándoles un nuevo órgano social estético y funcional. A pesar de los resultados prometedores, el ACV sigue teniendo un rendimiento inferior debido a las comorbilidades de inmunosupresión de por vida y las complicaciones infecciosas. La rata es un modelo animal ideal para estudios in vivo que investigan vías inmunológicas y mecanismos de rechazo del injerto. Las ratas también se utilizan ampliamente en nuevas técnicas de preservación de injertos de tejido compuesto, incluidos los estudios de perfusión y criopreservación. Los modelos utilizados para el ACV en ratas deben ser reproducibles, fiables y eficientes con baja morbilidad y mortalidad postoperatoria. Los procedimientos de trasplante de extremidades heterotópicas cumplen con estos criterios y son más fáciles de realizar que los trasplantes de extremidades ortotópicas. Dominar los modelos microquirúrgicos de roedores requiere una sólida experiencia en microcirugía y cuidado de animales. Aquí se informa un modelo confiable y reproducible de trasplante parcial de colgajo osteomiocutáneo heterotópico en ratas, los resultados postoperatorios y los medios de prevención de posibles complicaciones.

En este estudio de operador único, se realizaron 30 trasplantes parciales de extremidades heterotópicas singénicas. El éxito se definió en el día 21 postoperatorio como la ausencia de fracaso del ACV o complicaciones que requieren eutanasia. La evolución normal del injerto se representa en la Figura 3. La duración media de la obtención parcial de la extremidad y el recuadro del injerto en el receptor fue de 35 y 105 min, respectivamente; el tiempo medio de isquemia fue de 105 min. D...

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

Partial Heterotopic Hindlimb Transplantation Model in Rats

Este trabajo presenta un protocolo de trasplante de colgajo osteomiocutáneo heterotópico parcial en ratas y sus posibles resultados en el seguimiento a medio plazo.

Modelo de trasplante parcial de extremidades posteriores heterotópicas en ratas

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

Presentamos un modelo quirúrgico fiable y reproducible de trasplante heterotópico de extremidades posteriores en ratas. Esta técnica es ideal para estudios a corto plazo sobre preservación o rechazo inmune en VCA. La descripción paso a paso permitirá a los aprendices dominar rápidamente la cirugía.Para comenzar, haga una incisión circunferencial en la piel por encima del tobillo en el tercio distal de la pierna. Esqueletizar y cauterizar la arteria safena y la rama terminal de la arteria poplítea utilizando fórceps bipolares. Cauterizar y cortar los músculos gastrocnemio, sóleo, tibial anterior y bíceps femoral hasta que el hueso tibial quede expuesto.Haga una incisión de 2,5 centímetros en el pliegue inguinal derecho y diseccione la almohadilla de grasa inguinal. Use un retractor de anzuelo de pescado para agarrar el ligamento inguinal. Sostenga la almohadilla de grasa inguinal distalmente con pinzas de sujeción y retraiga la almohadilla de grasa para exponer los vasos femorales.Diseccionar los vasos femorales, individualizar las ramas de Murphy y ligar con lazos de nylon 8/0. Heparinizar la rata donante inyectando la heparina en la vena dorsal del pene con una aguja de calibre 27,5. Complete la incisión en la piel alrededor de la cadera.Use los fórceps bipolares para cauterizar los músculos bíceps femoral y glúteo superficial, luego cauterice y corte el nervio ciático a la mitad de la longitud del fémur. Exponer el fémur proximalmente a nivel de la cresta femoral posterior y ligar los vasos femorales con lazos de nylon a nivel del ligamento inguinal. Realice una arteriotomía en la arteria femoral justo debajo de la ligadura y dilate para insertar el angiocatéter de calibre 24.Cauterizar y cortar el músculo restante debajo del pedículo para exponer el lado anterior del fémur. Use el cortador de huesos para cortar la tibia proximalmente y cortar el fémur lo más distalmente posible. Enjuague la extremidad posterior parcial con dos mililitros de solución salina de heparina para obtener una salida venosa clara.Coloque la extremidad posterior sobre hielo en una gasa estéril hasta la transferencia microvascular. Diseccionar la almohadilla de grasa inguinal y reclinar la almohadilla de grasa inguinal distalmente para exponer los vasos femorales. Use un gancho para retraer el ligamento inguinal y pinzas de sujeción para sostener la almohadilla de grasa inguinal distalmente.Diseccionar los vasos femorales, individualizar las ramas de Murphy y ligar con lazos de nylon como se ha demostrado. Ligar ambos vasos por encima de los vasos epigástricos usando lazos de nylon. Coloque las abrazaderas del aproximador proximalmente, dilate la lente del vaso y enjuague con solución salina de heparina.Haga una incisión en el flanco izquierdo por encima de la cadera y cree una bolsa subcutánea con un túnel subcutáneo hasta el pliegue inguinal. Coloque la parte proximal de la extremidad parcial y la almohadilla de grasa inguinal a través del túnel subcutáneo para la transferencia microvascular. Realizar las anastomosis venosas y arteriales utilizando suturas de nylon.Retire ambas pinzas de aproximación y observe la revascularización de la extremidad. Realizar una prueba de ordeño en ambos vasos para evaluar la permeabilidad de cada anastomosis. Haga una incisión longitudinal en la piel en el lado medial de la extremidad trasplantada e inserte el injerto.Retire el exceso de piel del injerto y cierre la herida con suturas separadas y una sutura corriente utilizando suturas absorbibles 4/0. Suturar juntos las almohadillas de grasa inguinal de la extremidad trasplantada y el receptor usando dos suturas absorbibles separadas y cerrar el pliegue inguinal después del último chequeo de la anastomosis microvascular. Se monitorizó la evolución del modelo heterotópico de extremidades posteriores hasta el final del estudio.El recrecimiento del cabello se observó durante la primera semana postoperatoria, y la retracción cutánea apareció después de dos semanas. La falla del alotrasplante compuesto vascularizado puede ocurrir durante la primera semana postoperatoria debido a la trombosis microvascular. La trombosis venosa fue la causa de la eutanasia temprana en el 20% de los casos, todos los cuales ocurrieron antes del quinto día postoperatorio.La piel parecía azul y se oscurecía cada día. La automutilación es una preocupación seria en los injertos sin sentido y a menudo ocurre entre el segundo y el siete días postoperatorios. Si se limita a menos de un tercio de la superficie del injerto y se refiere solo a la piel, el desbridamiento quirúrgico y la sutura con suturas no absorbibles se pueden discutir con el veterinario del personal.La prevención de la automutilación se basa en el uso de un collar electrónico cosido en el cuello hasta el séptimo día postoperatorio y la limpieza de cualquier sangre o costra en las heridas quirúrgicas del animal. La autofagia severa de múltiples capas del injerto conduce a la eutanasia del animal. Los quistes dérmicos aparecieron después del día 14 postoperatorio, a veces con un centro necrótico cutáneo antes de la fístula.Es crucial asegurarse de que la colocación del injerto no impida que el animal sea ambulatorio, y que el pedículo no esté torcido o apretado durante el recuadro del injerto. Nuestro equipo realizó estudios de preservación en las extremidades adquiridas antes del trasplante. Se han evaluado los efectos de diferentes duraciones del almacenamiento en frío estático, así como diferentes protocolos de perfusión de extremidades.

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.

Modelo murino de CD40-activación de las células B

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

Investigación

Inmunología e Infección

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.

Ortotópico patas traseras trasplante en ratas

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 método de imagen para distinguir la inflamación aguda y crónica

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.

Crónica a largo plazo<em&gt; Pseudomonas aeruginosa</em&gt; Infección de las vías respiratorias en los ratones

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 e In Vivo la evaluación de la actividad supresora T, B y las células mieloides y las respuestas humorales de los receptores de trasplante

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.

Un modelo de derivación cardiopulmonar de recuperación sin transfusión o agentes inotrópicos en ratas

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.

Un modelo inmunológico para el trasplante heterotópico de células musculares cardíacas y cardíacas en ratas

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.

Optimización de la Técnica del Manguito para el Trasplante cardíaco murino

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.

Procedimientos de trasplante de médula ósea en ratones para estudiar la hematopoyesis clonal

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.

Purificación y expansión de células T asesinas naturales invariantes de ratón para estudios in vitro e in vivo

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