Reconocemos los Dres. Camillo Ricordi, Pileggi Antonello, R. Damaris Molano, Speier Esteban y Daniel Nyqvist para un debate fructífero. También damos las gracias Eleut Diego Hernández y Espinosa-Heidmann de asistencia técnica, y Mike Valdes y Formoso Margaret ayuda con grabación de video. Byron Maldonado grabado, editado y producido el vídeo final. Apoyo a la investigación fue proporcionada por el Instituto de Investigación de Diabetes Foundation ( <a href="http://www.DiabetesResearch.org" target="_blank">www.DiabetesResearch.org</a> ), el NIH / NIDDK / NIAID (F32DK083226 a MHA, NIH RO3DK075487 a AC; U01DK089538 a PO.B.). Apoyo a la investigación adicional para PO.B se proporciona a través de los fondos del Instituto Karolinska, el sueco Consejo de Investigación, la Fundación para la Diabetes sueca, la Fundación de la Familia Erling Persson-, la Familia Knut y Alice Wallenberg Foundation, el Skandia Insurance Company Ltd., vibrante ( FP7-228933-2), Programa Estratégico de Investigación en Diabetes en el Karolinska Inst.itutet, la Fundación Novo Nordisk, y la Fundación de amarre von Kantzow.

El siguiente procedimiento se realiza bajo el estereoscopio en 2 pasos, el primer paso consiste en cargar los islotes en la cánula y el segundo paso es el trasplante real en la ACE. Todos los procedimientos realizados con animales fueron aprobados por el Cuidado de Animales institucional y el empleo Comisión (IACUC) de la Universidad de Miami. 1. Islotes de carga en Cánula para trasplante <ol><li> Centro de los islotes en placa de cultivo al girar el plato en los círculos de estrechamiento. </li><li> Desconectar la cánula de la &quot;reserva&quot; y colocar la cánula y el tubo de conexión sobre una superficie limpia. El depósito puede ser hecho de una punta desechable 300 l pipeta de plástico sin filtro (Figura 1a). </li><li> Lavar las burbujas de aire fuera del depósito para asegurar flujo continuo de islotes cuando aspiración en el depósito. Enjuagar el depósito se hace por impulsar el manos libres motorizado jeringa controlador utilizando el pedal(Figura 1b, c). Esto también hará que el espacio en la jeringa para permitir la aspiración de los islotes en el depósito (pre-cargado con una solución estéril, tal como medios de solución salina, PBS o cultivo). </li><li> Aspirar suavemente cantidad deseada de islotes en el depósito. Islotes tenderá a girar al entrar en el depósito y se mantendrá juntos hacia la parte inferior. La aspiración se realiza por conducción hacia atrás la jeringa motorizada-conductor utilizando el pedal de pie. </li><li> Vuelva a conectar la cánula al depósito a través de la tubería de conexión. </li><li> Colocar la punta de la cánula de nuevo en la placa de cultivo y lavar los islotes fuera del depósito en el tubo a continuación, en la cánula. Asegúrese de que los islotes permanecer juntos como hace una copia de llenar el tubo / cánula con cuidado &quot;parpadeo&quot; (pulsar) del tubo (Figura 1d). Detener ya sea antes o después de que todas las burbujas de aire por delante de los islotes son expulsadas de la cánula. Si no está seguro, deje de islotes como entrar en la parte posterior de la cánula como el aire restanteburbujas antes de islotes puede ayudar a prevenir el reflujo (regurgitación) de los islotes de la ACE. Se disipará durante la noche. </li><li> En este punto, usted está listo para inyectar los islotes en el ACE (por favor, consulte los pasos siguientes). </li></ol> 2. El trasplante de islotes en la cámara anterior del ojo <ol><li> Coloque el ratón anestesiado sobre una almohadilla caliente bajo estereoscopio. </li><li> Coloque el hocico del ratón en anestesia &quot;máscara&quot; conectado a oxígeno / máquina de anestesia con isoflurano. La máscara está hecha de un plástico desechable de 1 ml punta de la pipeta (sin filtro) y conectado a un tubo de anestesia a través del extremo estrecho (figura 2a, b). </li><li> Retraer suavemente los párpados del ojo para ser trasplantados con el dedo índice y el pulgar de la mano libre y el &quot;pop&quot; el ojo para una mejor exposición y de fácil acceso (Figura 2c). Esto requerirá un poco de práctica para perfeccionar sin impedir la respiración del ratón por la presión excesiva en elcuello o bloqueando el flujo de sangre a la cabeza. </li><li> Usando una jeringuilla de insulina desechable (29 - 31 g) como bisturí, cuidadosamente penetrar sólo la punta en la córnea y hacer una incisión lateral única. Hacer la incisión en el punto medio entre el vértice de la córnea y el limbo para minimizar el reflujo de los islotes durante la inyección de la ACE (Figura 2d). </li><li> Cuidadosamente inserte la cánula (precargado con islotes) a través de la incisión. </li><li> Lentamente expulsar islotes fuera de la cánula y los depositan en la parte superior del iris. Para evitar el reflujo islote debido a la acumulación de presión excesiva en la ACE, expulsar los islotes en impulsos breves en tan pequeño volumen (s) como sea posible en el cuadrante opuesto a la incisión. Esto puede ser aún más asegurada mediante la compactación de islotes en el tubo durante la carga de la cánula (por favor, véase el paso 1,6). </li><li> Poco a poco retraer la cánula de la ACE. Este es un paso crítico, especialmente, si un gran volumen de islotes se inyectó como reflujo islote debido a la presenciaque se acumulan dentro de la ACE puede ser inevitable. Para eliminar / minimizar el reflujo islote, gire suavemente la cánula mientras que dentro de la ACE para liberar el exceso de presión a través de la incisión alrededor de la cánula. Busque signos de reflujo en tu intento de retirar la cánula y, si es necesario, espere hasta que desaparezca la presión antes de retraer completamente la cánula de la ACE. </li><li> Enjuagar el ojo trasplantado con PBS estéril o solución salina. </li><li> Inyectar buprenorfina para la analgesia postoperatoria (0.05 a 0.1 mg / kg, por vía subcutánea) para el primero 48 hr. </li><li> Aplique un ungüento antibiótico eritromicina oftálmica al ojo trasplantado inmediatamente después del trasplante. </li><li> Coloque de nuevo el animal en una jaula caliente para permitir la recuperación de la anestesia. </li></ol>

<ol>
	<li>Weigert, R., Sramkova, M., Parente, L., Amornphimoltham, P., Masedunskas, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+microscopy:+a+novel+tool+to+study+cell+biology+in+living+animals.">Intravital microscopy: a novel tool to study cell biology in living animals.</a> Histochem. Cell Biol. 133 (5), 481-491 (2010).</li><li>Leibiger, I. B., Caicedo, A., Berggren, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+in+vivo+imaging+of+pancreatic+?-cell+function+and+survival+-+a+perspective.">Non-invasive in vivo imaging of pancreatic ?-cell function and survival - a perspective.</a> Acta Physiol. (Oxf). , (2011).</li><li>Wang, Y., Maslov, K., Kim, C., Hu, S., Wang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+photoacoustic+and+fluorescence+confocal+microscopy.">Integrated photoacoustic and fluorescence confocal microscopy.</a> IEEE Trans Biomed. Eng. 57 (10), 2576-2578 (2010).</li><li>Ntziachristos, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Going+deeper+than+microscopy:+the+optical+imaging+frontier+in+biology.">Going deeper than microscopy: the optical imaging frontier in biology.</a> Nat. Methods. 7, 603-614 (2010).</li><li>Aswathy, R. G., Yoshida, Y., Maekawa, T., Kumar, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near-infrared+quantum+dots+for+deep+tissue+imaging.">Near-infrared quantum dots for deep tissue imaging.</a> Anal. Bioanal Chem. 397 (4), 1417-1435 (2010).</li><li>Ghoroghchian, P. P., Therien, M. J., Hammer, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+fluorescence+imaging:+a+personal+perspective.">In vivo fluorescence imaging: a personal perspective.</a> Wiley Interdiscip Rev. Nanomed Nanobiotechnol. 1 (2), 156-167 (2009).</li><li>Prescher, A., Mory, C., Martin, M., Fiedler, M., Uhlmann, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+FTY720+treatment+on+postischemic+pancreatic+microhemodynamics.">Effect of FTY720 treatment on postischemic pancreatic microhemodynamics.</a> Transplant Proc. 42 (10), 3984-3985 (2010).</li><li>Leblond, F., Davis, S., Vald&#233;s, P., Pogue, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-clinical+whole-body+fluorescence+imaging:+Review+of+instruments,+methods+and+applications.">Pre-clinical whole-body fluorescence imaging: Review of instruments, methods and applications.</a> J. Photochem. Photobiol. B. 98 (1), 77-94 (2010).</li><li>Toso, C., Vallee, J. P., Morel, P., Ris, F., Demuylder-Mischler, S., Lepetit-Coiffe, 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=Clinical+magnetic+resonance+imaging+of+pancreatic+islet+grafts+after+iron+nanoparticle+labeling.">Clinical magnetic resonance imaging of pancreatic islet grafts after iron nanoparticle labeling.</a> Am. J. Transplant. 8 (3), 701-706 (2008).</li><li>Denk, W., Strickler, J. H., Webb, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+laser+scanning+fluorescence+microscopy.">Two-photon laser scanning fluorescence microscopy.</a> Science. 248 (4951), 73-76 (1990).</li><li>Wang, B. G., Konig, K., Halbhuber, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+microscopy+of+deep+intravital+tissues+and+its+merits+in+clinical+research.">Two-photon microscopy of deep intravital tissues and its merits in clinical research.</a> J. Microsc. 238 (1), 1-20 (2010).</li><li>Denk, W., Delaney, K. R., Gelperin, A., Kleinfeld, D., Strowbridge, B. W., Tank, D. W., 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=Anatomical+and+functional+imaging+of+neurons+using+2-photon+laser+scanning+microscopy.">Anatomical and functional imaging of neurons using 2-photon laser scanning microscopy.</a> J. Neurosci. Methods. 54 (2), 151-162 (1994).</li><li>Cahalan, M. D., Parker, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choreography+of+cell+motility+and+interaction+dynamics+imaged+by+two-photon+microscopy+in+lymphoid+organs.">Choreography of cell motility and interaction dynamics imaged by two-photon microscopy in lymphoid organs.</a> Annu. Rev. Immunol. 26, 585-626 (2008).</li><li>Khorshidi, M. A., Vanherberghen, B., Kowalewski, J. M., Garrod, K. R., Lindstrom, S., Andersson-Svahn, H., 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=Analysis+of+transient+migration+behavior+of+natural+killer+cells+imaged+in+situ+and+in+vitro.">Analysis of transient migration behavior of natural killer cells imaged in situ and in vitro.</a> Integr. Biol. (Camb). 3 (7), 770-778 (2011).</li><li>Matheu, M. P., Cahalan, M. D., Parker, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoimaging:+studying+immune+system+dynamics+using+two-photon+microscopy.">Immunoimaging: studying immune system dynamics using two-photon microscopy.</a> Cold Spring Harb. Protoc. 2011, pdb.top99 (2011).</li><li>Celli, S., Albert, M. L., Bousso, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+the+innate+and+adaptive+immune+responses+underlying+allograft+rejection+by+two-photon+microscopy.">Visualizing the innate and adaptive immune responses underlying allograft rejection by two-photon microscopy.</a> Nat. Med. , (2011).</li><li>Fan, Z., Spencer, J., Lu, Y., Pitsillides, C., Singh, G., Kim, P., 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=In+vivo+tracking+of+'color-coded'+effector,+natural+and+induced+regulatory+T+cells+in+the+allograft+response.">In vivo tracking of 'color-coded' effector, natural and induced regulatory T cells in the allograft response.</a> Nat. Med. 16 (6), 718-722 (2010).</li><li>Sabek, O., Gaber, M. W., Wilson, C. M., Zawaski, J. A., Fraga, D. W., Gaber, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+human+islet+vascularization+using+a+dorsal+window+model.">Imaging of human islet vascularization using a dorsal window model.</a> Transplant Proc. 42 (6), 2112-2114 (2010).</li><li>Coppieters, K., Martinic, M. M., Kiosses, W. B., Amirian, N., von Herrath, M. <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+technique+for+the+in+vivo+imaging+of+autoimmune+diabetes+development+in+the+pancreas+by+two-photon+microscopy.">A novel technique for the in vivo imaging of autoimmune diabetes development in the pancreas by two-photon microscopy.</a> PLoS One. 5 (12), e15732 (2010).</li><li>Martinic, M. M., von Herrath, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+imaging+of+the+pancreas+during+development+of+diabetes.">Real-time imaging of the pancreas during development of diabetes.</a> Immunol Rev. 221, 200-213 (2008).</li><li>Mostany, R., Portera-Cailliau, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Method+for+2-Photon+Imaging+of+Blood+Flow+in+the+Neocortex+through+a+Cranial+Window.">A Method for 2-Photon Imaging of Blood Flow in the Neocortex through a Cranial Window.</a> J. Vis. Exp. (12), e678 (2008).</li><li>Palmer, G. M., Fontanella, A. N., Shan, S., Hanna, G., Zhang, G., Fraser, 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=In+vivo+optical+molecular+imaging+and+analysis+in+mice+using+dorsal+window+chamber+models+applied+to+hypoxia,+vasculature+and+fluorescent.">In vivo optical molecular imaging and analysis in mice using dorsal window chamber models applied to hypoxia, vasculature and fluorescent.</a> 6 (9), 1355-1366 (2011).</li><li>Jain, R. K., Munn, L. L., Fukumura, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+tumour+pathophysiology+using+intravital+microscopy.">Dissecting tumour pathophysiology using intravital microscopy.</a> Nat. Rev. Cancer. 2 (4), 266-276 (2002).</li><li>Taylor, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+response+of+capillary+endothelium+to+changes+in+intravascular+pressure,+as+seen+in+the+rabbit's+ear+chamber.">The response of capillary endothelium to changes in intravascular pressure, as seen in the rabbit's ear chamber.</a> Aust. J. Exp. Biol. Med. Sci. 31 (5), 533-543 (1953).</li><li>Shan, S., Sorg, B., Dewhirst, M. W. <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+mammary+window+of+orthotopic+breast+cancer+for+intravital+microscopy.">A novel rodent mammary window of orthotopic breast cancer for intravital microscopy.</a> Microvasc. Res. 65 (2), 109-117 (2003).</li><li>Speier, S., Nyqvist, D., Cabrera, O., Yu, J., Molano, R. D., Pileggi, 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=Noninvasive+in+vivo+imaging+of+pancreatic+islet+cell+biology.">Noninvasive in vivo imaging of pancreatic islet cell biology.</a> Nat. Med. 14 (5), 574-578 (2008).</li><li>Speier, S., Nyqvist, D., Kohler, M., Caicedo, A., Leibiger, I. B., Berggren, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+high-resolution+in+vivo+imaging+of+cell+biology+in+the+anterior+chamber+of+the+mouse+eye.">Noninvasive high-resolution in vivo imaging of cell biology in the anterior chamber of the mouse eye.</a> Nat. Protoc. 3 (8), 1278-1286 (2008).</li><li>Falck, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site+of+production+of+oestrogen+in+the+ovary+of+the+rat.">Site of production of oestrogen in the ovary of the rat.</a> Nature. 184, 1082 (1959).</li><li>Bickford-Wimer, P., Granholm, A. C., Bygdeman, M., Hoffer, B., Olson, L., Seiger, 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=Human+fetal+cerebellar+and+cortical+tissue+transplanted+to+the+anterior+eye+chamber+of+athymic+rats:+electrophysiological+and+structural+studies.">Human fetal cerebellar and cortical tissue transplanted to the anterior eye chamber of athymic rats: electrophysiological and structural studies.</a> Proc. Natl. Acad. Sci. U.S.A. 84 (16), 5957-5961 (1987).</li><li>Adeghate, E., Donath, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+findings+in+long-term+pancreatic+tissue+transplants+in+the+anterior+eye+chamber+of+rats.">Morphological findings in long-term pancreatic tissue transplants in the anterior eye chamber of rats.</a> Pancreas. 5 (3), 298-305 (1990).</li><li>Abdulreda, M. H., Faleo, G., Molano, R. D., Lopez-Cabezas, M., Molina, J., Tan, 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=High-resolution,+noninvasive+longitudinal+live+imaging+of+immune+responses.">High-resolution, noninvasive longitudinal live imaging of immune responses.</a> Proc. Natl. Acad. Sci. U.S.A. , (2011).</li><li>Unutmaz, D., Xiang, W., Sunshine, M. J., Campbell, J., Butcher, E., Littman, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+primate+lentiviral+receptor+Bonzo/STRL33+is+coordinately+regulated+with+CCR5+and+its+expression+pattern+is+conserved+between+human+and+mouse.">The primate lentiviral receptor Bonzo/STRL33 is coordinately regulated with CCR5 and its expression pattern is conserved between human and mouse.</a> J. Immunol. 165 (6), 3284-3292 (2000).</li><li>Pileggi, A., Molano, R. D., Berney, T., Cattan, P., Vizzardelli, C., Oliver, 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=Heme+oxygenase-1+induction+in+islet+cells+results+in+protection+from+apoptosis+and+improved+in+vivo+function+after+transplantation.">Heme oxygenase-1 induction in islet cells results in protection from apoptosis and improved in vivo function after transplantation.</a> Diabetes. 50 (9), 1983-1991 (2001).</li></ol>

PO.B. Es uno de los fundadores de la empresa de biotecnología BioCrine, que se va a utilizar la cámara anterior del ojo como una plataforma de servicio comercial. AC se encuentra en la patente que protege esta tecnología.

Islotes pancreáticos de murino fueron aisladas utilizando digestión con colagenasa seguido por purificación en gradientes de densidad, como se ha descrito previamente 33. Islotes aislados se cultivaron durante la noche antes del trasplante. Si bien esto puede no ser necesario, se recomienda para permitir que los islotes de recuperarse del procedimiento de aislamiento. Esto es crítico cuando se realiza el trasplante en los receptores diabéticos, ya que garantizará el trasplante de islotes sobrevivientes ...

Los avances en microscopía intravital han revelado fenómenos fisiológicos no predichas por los estudios in vitro 1. Esto pone de relieve el desafío en la traducción de los resultados obtenidos por los métodos in vitro convencional en el animal vivo. En la última década, la visualización de los tejidos en los animales vivos se mejoró considerablemente por los avances tecnológicos en las modalidades de imagen 2, 3, 4, 5, 6. Esto ha estimulado la necesidad de enfoques en formación de imágenes in vivo con aplicación factible en modelos animales experimentales para permitir la visualización longitudinal de los tejidos diana de manera no invasiva. Las técnicas de imagen como la resonancia magnética y la tomografía por emisión de positrones o bioluminiscencia han permitido que la imagen no invasiva de órganos / tejidos profundos dentro del cuerpo 7-8, 9. Pero estas técnicas no pueden lograr células únicas resolución debido a señales de fondo elevadas y baja resolución espacial, a pesar del uso of materiales de alto contraste o 4 tejidos específicos de luminiscencia. Esto fue tratada con la llegada de dos fotones microscopía de fluorescencia confocal 10. Dos fotones microscopía intravital permitió estudios de formación de imágenes para visualizar y cuantificar acontecimientos celulares con detalles sin precedentes 11, 12. Esto ha llevado a la caracterización de procesos biológicos esenciales en la salud y la enfermedad 13, 14, 15, 16. Mientras pioneros estudios de imagen han intravital principalmente &quot;imitado&quot; en condiciones in vivo en los tejidos extirpados (ganglios linfáticos, por ejemplo), otros estudios han utilizado métodos invasivos para los tejidos diana de imagen expuestos in situ 17, 18, ​​19, 20, 21. Otros estudios también han utilizado modelos de cámara &quot;ventana&quot; para eludir las limitaciones asociadas con los métodos invasivos y resolución de imágenes limitado en vivo 22, 23, 24, 25. En el modelo de cámara de ventana, una cámara con una ventana transparente se implanta quirúrgicamente en la piel en difeubicaciones de alquiler (piel dorsal o el oído, almohadilla de grasa mamaria, hígado, etc) sobre el animal (por ejemplo, ratón, rata, conejo). Si bien este enfoque claramente permite una alta resolución de imágenes in vivo, se requiere una cirugía invasiva para implantar la cámara y no puede ser capaz de acomodar los estudios longitudinales de imagen durante varias semanas o meses 22. Se ha demostrado recientemente que la combinación de alta resolución de la microscopía confocal con un procedimiento mínimamente invasivo, es decir, el trasplante a la cámara anterior del ojo (ACE) proporciona una &quot;ventana cuerpo natural&quot; como un. Potente y versátil en vivo plataforma de imágenes 26, 27 El trasplante en el ACE se ha utilizado en las últimas décadas para estudiar aspectos biológicos de una variedad de tejidos 28, 29, 30, y su combinación reciente con imágenes de alta resolución permitió el estudio de la fisiología de los islotes pancreáticos con células únicas resolución no invasiva y longitudinalmente &lt;sup&gt; 26, 27. Este enfoque se utilizó para estudiar las respuestas autoinmunes durante el desarrollo de la diabetes tipo 1 en modelos animales (datos no publicados). También fue utilizado para estudiar el desarrollo de páncreas, así como, en los estudios de la función renal mediante el trasplante en las yemas de páncreas ECA o individuales glomérulos renales, respectivamente (datos no publicados). Un reciente informe utilizando este enfoque demostró además su solicitud para estudiar la respuesta inmune después de trasplante de islotes pancreáticos 31. Es importante destacar que, este estudio demostró que el trasplante a la cámara anterior del ojo proporciona una ventana natural del cuerpo para llevar a cabo: (1) longitudinal, la imagen no invasiva de tejidos trasplantados in vivo, (2) in vivo cytolabeling para evaluar el fenotipo celular y la viabilidad en situ, (3) seguimiento en tiempo real de la infiltración de células inmunes en el tejido diana, y (4) la intervención local por aplicación tópica o inyección intraocular. En este sentido, demonstrate cómo realizar el trasplante en la cámara anterior del ojo utilizando islotes pancreáticos.

<table border="1"><tbody><tr><td> Nombre de reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Descripción / Comentarios </td></tr><tr><td> IsoTHESIA (isoflurano) </td><td> Buttler Animal Health Supply </td><td> 11695-6775-2 </td><td> 99,9% de isoflurano / ml </td></tr><tr><td> Ketaset (Ketamina HCL) </td><td> Fort Dodge Animal Health </td><td> 0856-2013-01 </td><td> Anestesia inyectable Alternativa </td></tr><tr><td> Beprenex (buprenorfina HCL) </td><td> Reckitt Benckiser Health Care (UK) Ltd. </td><td> 12496-075-7-1 </td><td> 0,3 mg / ml </td></tr><tr><td> Ophthalmic eritromicina ungüento USP, 0,5% </td><td> Akron </td><td> 17478-070-35 </td><td> Aplicado preventivamente a ojo trasplantado </td></tr><tr><td> 0,9% de cloruro sódico (solución salina) </td><td> Hospira Inc. </td><td> 0409-7983-03 </td><td> Para la inyección iv. Estéril </td></tr><tr><td> PBS </td><td> Gibco </td><td> 10010-023 </td><td> 1X. Estéril </td></tr><tr><td> CMRL medio 1066 </td><td> Cellgro </td><td> 98-304 CV- </td><td> Complementado, CIT modificación. Los medios preferidos para islotes </td></tr></tbody></table>

transplantation into the anterior chamber of the eye for longitudinal, non-invasive in vivo imaging with single-cell resolution in real-time

Intravital de imágenes se ha convertido en una herramienta indispensable en la investigación biológica. En el proceso, muchas técnicas de imagen se han desarrollado para estudiar los diferentes procesos biológicos en los animales no invasiva. Sin embargo, una limitación técnica importante en existentes modalidades de imagen intravital es la incapacidad para combinar la no-invasivo, formación de imágenes longitudinal con capacidades de resolución de una sola célula. Se muestra aquí cómo el trasplante a la cámara anterior del ojo elude esta limitación significativa que ofrece una plataforma versátil experimental que permite no invasiva, la imagen longitudinal con resolución celular in vivo. Se demuestra el procedimiento de trasplante en el ratón y proporcionar resultados representativos usando un modelo con relevancia clínica, es decir, trasplante de islotes pancreáticos. Además de permitir la visualización directa en una variedad de tejidos trasplantados en la cámara anterior del ojo, este enfoque proporciona una plataforma para Screen mediante la realización de las drogas a largo plazo de seguimiento y monitoreo en los tejidos diana. Debido a su versatilidad, el tejido / trasplante de células en la cámara anterior del ojo no sólo terapias de trasplante beneficios, se extiende a otras aplicaciones in vivo para estudiar los procesos fisiológicos y fisiopatológicos tales como la transducción de señales y el cáncer o desarrollo de la enfermedad autoinmune.

Hay algunos parámetros que definen una &quot;buena&quot; trasplante. Un trasplante de buena es la que procede sin sangrado cuando se hace la incisión como se puede ver en el vídeo. El sangrado se impide / minimiza al penetrar sólo la punta del escalpelo (aguja) en la ACE (Figura 3a). Esto también ayudará a evitar el contacto y la punción del iris. También se asegurará de una pequeña incisión que se curan muy bien sin causar opacidad de la córnea con el tiempo (Figura 3c, d)....

Watch this Scientific Journal Video about Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time at JoVE.com

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

Un nuevo enfoque de la combinación de trasplante intraocular y microscopía confocal permite longitudinal, no invasiva imágenes en tiempo real con una sola célula de resolución dentro de los tejidos injertados<em&gt; In vivo</em&gt;. Se demuestra cómo trasplantar islotes pancreáticos en la cámara anterior del ojo del ratón.

Trasplante en la cámara anterior del ojo para Longitudinal, no invasiva<em&gt; En vivo</em&gt; Imagen con una sola célula de resolución en tiempo real

Intravital imaging has emerged as an indispensable tool in biological research.  In the process, many imaging techniques have been developed to study ...

transplantation-into-anterior-chamber-eye-for-longitudinal-non

Research

JoVE Journal

Medicine

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

13.3K vistas.  University of Miami Miller School of Medicine. Un nuevo enfoque de la combinación de trasplante intraocular y microscopía confocal permite longitudinal, no invasiva imágenes en tiempo real con una sola célula de resolución dentro de los tejidos injertados In vivo. Se demuestra cómo trasplantar islotes pancreáticos en la cámara anterior del ojo del ratón.

Video: Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

High-frequency High-resolution Echocardiography: First Evidence on Non-invasive Repeated Measure of Myocardial Strain, Contractility, and Mitral Regurgitation in the Ischemia-reperfused Murine Heart

Ischemia-reperfusion (IR) was surgically performed in murine hearts which were then subjected to repeated imaging to monitor temporal changes in functional parameters of key clinical significance. Two-dimensional movies were acquired at high frame rate (8 kHz) and were utilized to estimate high-quality myocardial strain. Two-dimensional elastograms (strain images), as well as strain profiles, were visualized. Results were powerful in quantitatively assessing IR-induced changes in cardiac events including left-ventricular (LV) contraction, LV relaxation and isovolumetric phases of both pre-IR and post-IR beating hearts in intact mice. In addition, compromised sector-wise wall motion and anatomical deformation in the infarcted myocardium were visualized.  The elastograms were uniquely able to provide information on the following parameters in addition to standard physiological indices that are known to be affected by myocardial infarction in the mouse: internal diameters of mitral valve orifice and aorta, effective regurgitant orifice, myocardial strain (circumferential as well as radial), turbulence in blood flow pattern as revealed by the color Doppler movies and velocity profiles, asynchrony in LV sector, and changes in the length and direction of vectors demonstrating slower and asymmetrical wall movement. This work emphasizes on the visual demonstration of how such analyses are performed. 

High-frequency High-resolution Echocardiography: First Evidence on Non-invasive Repeated Measure of Myocardial Strain, Contractility, and Mitral Regurgitation in the Ischemia-reperfused Murine Heart 

High frequency Doppler ultrasound is a novel technology for assessing regional myocardial function. This work presents first evidence demonstrating applicability of this versatile imaging platform for the repeated measure of myocardial strain, dp/dt, and mitral regurgitation in the ischemia-reperfused (IR) murine heart.

Alta frecuencia de alta resolución Ecocardiografía: Primera evidencia sobre la no-invasivo medida repetida de la tensión de miocardio, la contractilidad y la insuficiencia mitral en el corazón por isquemia-reperfusión murino

Ischemia-reperfusion (IR) was surgically performed in murine hearts which were then subjected to repeated imaging to monitor temporal changes in ...

Investigación

Medicina

Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical excision of the tumor with a full lymphadenectomy in the drainage area of the tumor. However, removal of lymph nodes is associated with increased morbidity due to infection, wound breakdown and lymphedema.4,5 As an alternative, the sentinel lymph node procedure (SLN) was developed several decades ago to detect the first draining lymph node from the tumor.6 In case of lymphogenic dissemination, the SLN is the first lymph node that is affected (Figure 1). Hence, if the SLN does not contain metastases, downstream lymph nodes will also be free from tumor metastases and need not to be removed. The SLN procedure is part of the treatment for many tumor types, like breast cancer and melanoma, but also for cancer of the vulva and cervix.7 The current standard methodology for SLN-detection is by peritumoral injection of radiocolloid one day prior to surgery, and a colored dye intraoperatively. Disadvantages of the procedure in cervical and vulvar cancer are multiple injections in the genital area, leading to increased psychological distress for the patient, and the use of radioactive colloid.
Multispectral fluorescence imaging is an emerging imaging modality that can be applied intraoperatively without the need for injection of radiocolloid. For intraoperative fluorescence imaging, two components are needed: a fluorescent agent and a quantitative optical system for intraoperative imaging. As a fluorophore we have used indocyanine green (ICG). ICG has been used for many decades to assess cardiac function, cerebral perfusion and liver perfusion.8 It is an inert drug with a safe pharmaco-biological profile. When excited at around 750 nm, it emits light in the near-infrared spectrum around 800 nm. A custom-made multispectral fluorescence imaging camera system was used.9.
The aim of this video article is to demonstrate the detection of the SLN using intraoperative fluorescence imaging in patients with cervical and vulvar cancer. Fluorescence imaging is used in conjunction with the standard procedure, consisting of radiocolloid and a blue dye. In the future, intraoperative fluorescence imaging might replace the current method and is also easily transferable to other indications like breast cancer and melanoma.

Fluorescence imaging is a promising innovative modality for image-guided surgery in surgical oncology. In this video we describe the technical procedure for detection of the sentinel lymph node using fluorescence imaging as showcased in gynecologic oncologicy. A multispectral fluorescence camera system, together with the fluorescent agent indocyanine green, is applied.

Multiespectrales en tiempo real imágenes de fluorescencia para la detección intraoperatoria del ganglio centinela en Oncología Ginecológica

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical ...

Non-invasive Imaging of Acute Allograft Rejection after Rat Renal Transplantation Using 18F-FDG PET

The number of patients with end-stage renal disease, and the number of kidney allograft recipients continuously increases. Episodes of acute cellular allograft rejection (AR) are a negative prognostic factor for long-term allograft survival, and its timely diagnosis is crucial for allograft function 1. At present, AR can only be definitely diagnosed by core-needle biopsy, which, as an invasive method, bares significant risk of graft injury or even loss. Moreover, biopsies are not feasible in patients taking anticoagulant drugs and the limited sampling site of this technique may result in false negative results if the AR is focal or patchy. As a consequence, this gave rise to an ongoing search for new AR detection methods, which often has to be done in animals including the use of various transplantation models.
Since the early 60s rat renal transplantation is a well-established experimental method for the examination and analysis of AR 2. We herein present in addition small animal positron emission tomography (PET) using 18F-fluorodeoxyglucose (FDG) to assess AR in an allogeneic uninephrectomized rat renal transplantation model and propose graft FDG-PET imaging as a new option for a non-invasive, specific and early diagnosis of AR also for the human situation 3. Further, this method can be applied for follow-up to improve monitoring of transplant rejection 4.

Non-invasive Imaging of Acute Allograft Rejection after Rat Renal Transplantation Using 18F-FDG PET

We herein present a rat renal transplantation model to non-invasively assess acute allograft rejection using positron emission tomography with 18F-fluorodeoxyglucose.

Imagen no invasiva de rechazo agudo del aloinjerto después Rat Trasplante Renal Uso<sup&gt; 18</sup&gt; F-FDG PET

The number of patients with end-stage renal  disease, and the number of kidney allograft recipients continuously increases.  Episodes of acute cellular ...

Development of an Audio-based Virtual Gaming Environment to Assist with Navigation Skills in the Blind

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind. Using only audio based cues and set within the context of a video game metaphor, users gather relevant spatial information regarding a building's layout. This allows the user to develop an accurate spatial cognitive map of a large-scale three-dimensional space that can be manipulated for the purposes of a real indoor navigation task. After game play, participants are then assessed on their ability to navigate within the target physical building represented in the game. Preliminary results suggest that early blind users were able to acquire relevant information regarding the spatial layout of a previously unfamiliar building as indexed by their performance on a series of navigation tasks. These tasks included path finding through the virtual and physical building, as well as a series of drop off tasks. We find that the immersive and highly interactive nature of the AbES software appears to greatly engage the blind user to actively explore the virtual environment. Applications of this approach may extend to larger populations of visually impaired individuals.

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind.

Desarrollo de un entorno de juego basado en audio virtual para ayudar con habilidades de navegación en la ciega

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind. Using only audio ...

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an increased risk of vascular morbidity.&#160;Yet, albeit&#160;the mounting clinical evidence, there is a paucity of clear studies of vascular toxicity and therefore the etiology of a heterogeneous group of vascular/cardiovascular disorders remains to be elucidated. Moreover, the mechanism that may underlie vascular toxicity can completely differ from the principles of chemotherapy-induced cardiotoxicity, which is related to direct myocyte injury. We have established a real-time, in vivo molecular imaging platform&#160;to evaluate&#160;the potential&#160;acute&#160;vascular toxicity of anti-cancer therapies.
We have set up a platform of in vivo, high-resolution molecular imaging&#160;in mice,&#160;suitable for visualizing vasculature within confined organs and reference blood vessels within the same individuals whereas each individual serve as its own control. Blood vessel walls were impaired after doxorubicin administration, representing a unique mechanism of vascular toxicity that may be the early event in end-organ injury. Herein, the method of fibered confocal fluorescent microscopy (FCFM) based imaging is described, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

We herein describe the method of fibered confocal fluorescent microscopy (FCFM) based imaging, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Inducida por la quimioterapia Vascular Toxicidad - En tiempo real<em&gt; En vivo</em&gt; Imagen de Deterioro del buque

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an ...

Non-invasive Assessment of the Efficacy of New Therapeutics for Intestinal Pathologies Using Serial Endoscopic Imaging of Live Mice

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic mechanisms that contribute to the onset and progression of intestinal diseases. The identification of new molecules that promote these pathologies has led to a flurry of activity focused on the development of potential new therapies to inhibit their function. As a result, various pre-clinical mouse models with an intact immune system and stromal microenvironment are now heavily used. Here we describe three experimental protocols to test the efficacy of new therapeutics in pre-clinical models of (1) acute mucosal damage, (2) chronic colitis and/or colitis-associated colon cancer, and (3) sporadic colorectal cancer. We also outline procedures for serial endoscopic examination that can be used to document the therapeutic response of an individual tumor and to monitor the health of individual mice. These protocols provide complementary experimental platforms to test the effectiveness of therapeutic compounds shown to be well tolerated by mice.

We describe methods for longitudinal monitoring of the efficacy of therapeutics for the treatment of colonic pathologies in mice using a rigid endoscope. This protocol can be readily used for the characterization of the therapeutic response of an individual tumor in live mice and also for monitoring potential disease relapse.

Evaluación no invasiva de la eficacia de nuevas terapias para Intestinal Patologías recurriendo a imágenes de Endoscópica serie de ratones vivos

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic ...

Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular weight metabolites in tissues of the treated host to achieve long-term survival. The semipermeable membrane allows engrafted encapsulated cells to avoid rejection by the immune system. The encapsulation procedure was designed to enable a controlled release of bioactive compounds, such as insulin, other hormones, and cytokines. Here we describe a method for encapsulation of catabolic cells, which consume lipids for heat production and energy dissipation (thermogenesis) in the intra-abdominal adipose tissue of obese mice. Encapsulation of thermogenic catabolic cells may be potentially applicable to the prevention and treatment of obesity and type 2 diabetes. Another potential application of catabolic cells may include detoxification from alcohols or other toxic metabolites and environmental pollutants.

Here, we present a protocol for encapsulation of catabolic cells, which consume lipids for heat production in intra-abdominal adipose tissue and increase energy dissipation in obese mice.

La encapsulación termogénico preadipocitos para el trasplante en los depósitos de tejido adiposo

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular ...

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment.

This protocol describes the use of multiphoton microscopy to perform extended time-lapse imaging of multicellular interactions in real time, in vivo at single cell resolution.

Prolongado lapso de tiempo intravital de imágenes de tiempo real multicelular Dinámica en el microambiente tumoral

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and ...

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular plasticity or damage occurring overtime and in relation to neuronal activity or blood flow. In vivo two-photon microscopy allows the study of the structural and functional plasticity of large cellular units in the living brain. In particular, the thinned-skull window preparation allows the visualization of cortical regions of interest (ROI) without inducing significant brain inflammation. Repetitive imaging sessions of cortical ROI are feasible, providing the characterization of disease hallmarks over time during the progression of numerous CNS diseases. This technique accessing the pial structures within 250 &#956;m of the brain relies on the detection of fluorescent probes encoded by genetic cellular markers and/or vital dyes. The latter (e.g., fluorescent dextrans) are used to map the luminal compartment of cerebrovascular structures. Germane to the protocol described herein is the use of an in vivo marker of amyloid deposits, Methoxy-O4, to assess Alzheimer&#39;s disease (AD) progression. We also describe the post-acquisition image processing used to track vascular changes and amyloid depositions. While focusing presently on a model of AD, the described protocol is relevant to other CNS disorders where pathological cerebrovascular changes occur.

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

This manuscript describes a procedure to track the remodeling of the cerebrovasculature during amyloid plaque accumulation in vivo using longitudinal two-photon microscopy. A thinned-skull preparation enables the visualization of fluorescent dyes to assess the progression of cerebrovascular damage in a mouse model of Alzheimer&#39;s disease.

Longitudinal<em&gt; En Vivo</em&gt; Obtención de imágenes de la vasculatura cerebral: Importancia de las enfermedades del sistema nervioso central

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular ...

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. To better understand the mechanism behind these surgical procedures, a rat partial liver transplantation (PLTx) model was established for relevant surgical studies. Because of the complexity of the rat PLTx model, a protocol with detailed descriptions is required. An article published previously reported a protocol in which ex vivo hepatectomy was used to achieve 50% rat PLTx. In contrast to this protocol, we introduced a re-arterialized PLTx with an in vivo 70% hepatectomy. An updated vessel-oriented hepatectomy was incorporated into the rat PLTx to refine the microsurgical procedure. The portal veins and hepatic arteries of the left lateral lobe and the median lobe were individually dissected and ligated before removal of the liver parenchyma, thereby decreasing the probability of bleeding in the remnant liver stump. Furthermore, an end-to-side vessel anastomosis between the common hepatic artery and the enlarged proper hepatic artery was introduced to re-arterialize the hepatic artery. By using this end-to-side vessel anastomosis technique, the diameter of the anastomosis was enlarged, thereby decreasing the difficulty of hand suture and maintaining a high rate of anastomotic patency. Moreover, the cuff anastomosis of the infrahepatic vena cava was slightly modified. A section of circumferential liver parenchyma around the vena cava of a recipient was preserved during cuff anastomosis to maintain the three-dimensional shape of the vascular lumen. This section of liver parenchyma was removed after completing the anastomosis. With this modification, the step involving placement of stay sutures was omitted, thereby further shortening the cuff anastomosis time. By using this protocol of rat PLTx, a low liver enzyme level, an intact liver lobular architecture and a high survival rate were achieved after microsurgery.

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Here, a protocol involving re-arterialized rat partial liver transplantation is presented. Specifically, 70% liver was resected in vivo by using an updated technique of vessel-oriented hepatectomy. The hepatic artery was reconstructed in an end-to-side manner. The cuff technique was modified to shorten the anastomosis time of the infrahepatic vena cava.

Volver a arterializada rata parcial trasplante del hígado con una en vivo orientada al barco 70% hepatectomía

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. ...