Los autores desean agradecer a Adriane Mosley y Libuse Jerabek (Stanford) para obtener ayuda con la técnica quirúrgica.

Todos los estudios con animales se realizaron de acuerdo con las directrices de cuidado de los animales y el uso comité de la UCLA y los Institutos Nacionales de Salud de Guía para el Cuidado y Uso de Animales de Laboratorio. La duración del procedimiento que se describe a continuación es de aproximadamente 45 a 60 minutos de principio a fin. 1. Preparación de campo quirúrgico <ol><li> Realice el procedimiento en una sala de cirugía animal limpio. </li><li> Equipamiento: isoflurano vaporizador, Gaymer Bomba T con almohadilla térmica. </li><li> Herramientas estériles: dos pinzas curvas, tijeras finas, porta agujas. </li><li> Guantes estériles se deben utilizar durante todo el procedimiento. </li></ol> 2. Preparación de los animales <ol><li> Coloque dos ratones machos o hembras, de mismo fondo genético, de peso y de tamaño similar en la misma jaula y controlar durante al menos dos semanas para asegurar la cohabitación armoniosa. Los ratones hembra que se prefieren debido a su comportamiento menos agresivo. </li><li> Anestesiar animales porusando un vaporizador de isoflurano. Coloque los ratones en una cámara de inducción Posi-Seal conectados al vaporizador de isoflurano (4-5% v / v). Una vez que la anestesia se induce, transferir el animal a la zona de afeitado de la piel y mantener la anestesia durante todo el procedimiento a través de un cono de nariz conectado a isoflurano (1,5-2% v / v). Aplique una pomada oftálmica con un Q-tip para evitar que los ojos secos. </li><li> Colocar el animal en posición supina. A fondo afeitar el lado izquierdo del ratón a la izquierda y el lado derecho del ratón colocado a la derecha a partir de aproximadamente 1 cm por encima del codo hasta 1 cm por debajo de la rodilla. </li><li> Asépticamente preparar las áreas afeitadas limpiando a fondo (2-3x) con toallitas Betadine empapado seguido de toallitas con alcohol. Coloque el ratón sobre una almohadilla climatizada cubierta por una gasa estéril. </li><li> Para la analgesia, carprofeno y administrar buprenorfina por vía intraperitoneal o por vía subcutánea a una dosis de 10 mg / kg y 0,1 mg / kg, respectivamente. </li><li> Coloca los animales de su lado, de espaldas, con undjacent áreas afeitadas hacia arriba. Para evitar cualquier tipo de contaminación de la zona quirúrgica, cubra los ratones con un paño estéril exponiendo sólo el área de operación. Crear una pequeña abertura drapeado permanecer estéril cuando se realiza la cirugía. Hicimos la ventana cortina grande como para tener una mejor visión durante la grabación en vídeo. </li></ol> 3. Parabiosis <ol><li> Uso de una tijera afilada, realizar incisiones en la piel longitudinales a los lados afeitado de cada partida animal en 0,5 cm por encima del codo hasta el final a 0,5 cm por debajo de la articulación de la rodilla (Figura 1). Tras la incisión, separe suavemente la piel de la fascia subcutánea mediante la celebración de la piel con un par de pinzas curvas y separar la fascia con un segundo par de crear 0,5 cm de piel libre. Lleve a cabo esta separación a lo largo de toda la incisión. </li><li> Comience la unión uniendo el olécranon izquierda de un animal a la olécranon derecho de la otra. Ambos olecranons y articulaciones de la rodilla son claramente distinguibles siguiente ªincisión en la piel e. Para facilitar la unión, doble el codo del primer ratón y pasar la aguja de la no absorbible 3-0 sutura bajo el olécranon. Del mismo modo, doblar el codo de la segunda ratón y pasar la misma sutura debajo de ella. Coloque las juntas firmemente por un nudo quirúrgico doble. </li><li> Conectar las articulaciones de la rodilla siguiendo el mismo procedimiento. </li><li> Después de la fijación de las articulaciones, conecte la piel de los dos animales con un absorbible 5-0 Vicryl sutura continua a partir ventralmente desde el codo hacia la rodilla. Para evitar la rotura de la piel y la separación realizar un cierre estanco de sutura de la piel en el área alrededor de los codos y las rodillas. Una vez que la fijación a la piel ventral ha sido completado, realizar un nudo quirúrgico doble. Coloque el ratón en la posición prona y continuar con el dorsal de sutura que termina con un nudo quirúrgico doble. Verificar la continuidad de la sutura y confirmar la falta de aberturas. </li><li> Administrar 0,5 ml de NaCl al 0,9% por vía subcutánea a cada ratón para evitar deshidratación. </li></ol> 4. La recuperación postoperatoria <ol><li> Mantener a los animales en el cojín calentado hasta su recuperación. </li><li> Después de la recuperación, proporcionar analgésicos carprofeno y buprenorfina. Repita por vía intraperitoneal o por vía subcutánea cada 24 y 12 h, respectivamente, durante 48 horas a las mismas dosis descritas anteriormente (paso 2.5). Observar a los animales en busca de signos de dolor y angustia como temblores, letargia, la masticación de la cola, se arqueó hacia atrás, la falta de preparación, etc al día durante dos semanas. </li><li> Profiláctica, el tratamiento de ratones con sulfametoxazol / trimetoprima suspensión oral en su botella de agua de 2 mg sulfa / ml 0.4 mg / ml recortar por 10 días para prevenir infecciones bacterianas. </li><li> Casa cada par parabiotic en una jaula limpia con material de cama monolítica (por ejemplo, una toalla de papel o una almohadilla absorbente estéril) para prevenir la aspiración del material de cama. Volver a los animales a una jaula de cama rellenos cuando son capaces de mantener decúbito esternal con la cabeza erguida. Para reducir al mínimo la cepade llegar a los alimentos mientras se ajusta a la existencia parabiotic, colocar las bolitas de comida humedecida en el suelo de la jaula. Proporcionar material de nidificación. En 1-2 semanas los ratones parabiotic tienen la capacidad para caminar normalmente delanteras y traseras-extremidades pares quirúrgicamente. </li><li> Quimerismo sangre ocurre 10-14 días después de la cirugía. </li></ol> 5. Confirmación y procedimiento inverso <ol><li> Después de dos semanas extraer la sangre de las venas de la cola de cada ratón para el análisis de citometría de flujo-para confirmar el quimerismo de la sangre (Figura 3). </li><li> Procesado de la sangre para el análisis de citometría de flujo: diluir 2-3 gotas de sangre en 500 l de EDTA 10 mM. Añadir 500 l de 2% de dextrano (diluido en PBS), mezclar bien y se incuba a 37 ° C durante 30 min. Transferir el sobrenadante en un nuevo tubo, centrifugar a 1200 rpm durante 5 min y resuspender las células sedimentadas en PBS (u otro tampón) para el análisis de citometría de flujo. </li><li> Dependiendo del diseño experimental, el par parabiotic se puede separar unat puntos de tiempo posteriores. En nuestra experiencia, hemos mantenido pares parabiosed para hasta 9 meses sin ningún tipo de complicaciones. </li><li> Realice el procedimiento de reversa en una superficie quirúrgico estéril. Anestesie ratones colocándolos en una cámara de inducción conectada a un vaporizador de isoflurano (4-5%) y mantener la anestesia con isoflurano 1,5-2%, como se describe anteriormente (paso 2.2). </li><li> Retire el cabello del área que rodea la sutura inicial y preparar asépticamente las áreas afeitadas como se describe anteriormente (pasos 2.3, 2.4). </li><li> Para la analgesia, carprofeno y administrar buprenorfina por vía intraperitoneal o por vía subcutánea a una dosis de 10 mg / kg y 0,1 mg / kg, respectivamente. </li><li> Cubra el área quirúrgica con un paño estéril como se describió anteriormente (paso 2.6). </li><li> Usando unas tijeras afiladas, separar los ratones a través de una incisión longitudinal a lo largo de la sutura lateral y separar suavemente la fascia recién formado entre los dos ratones con un par de pinzas curvas. Para separar las articulaciones, cortar los nudos de la suturaconectarlos. Se quita la piel a lo largo de la incisión para lograr bordes lisos y vuelva a colocar la piel con una continua absorbible 5-0 recubierto de sutura Vicryl. </li><li> Para prevenir la deshidratación, administrar 0,5 ml de NaCl al 0,9% por vía subcutánea a cada ratón. Mantener a los animales en el cojín calentado hasta su recuperación. </li><li> Después de la recuperación, repita la administración de analgésicos, carprofeno (10 mg / kg) cada 24 horas y la buprenorfina (0,1 mg / kg) cada 12 horas durante 48 horas. </li><li> Para prevenir las infecciones bacterianas tratar ratones con sulfametoxazol / trimetoprima suspensión oral (2 mg sulfa / ml 0.4 mg de modelo / ml) en su botella de agua durante 10 días. </li></ol>

<ol>
	<li>Bert, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exp&#233;riences+et+consid&#233;rations+sur+la+greffe+animale.">Exp&#233;riences et consid&#233;rations sur la greffe animale.</a> Journal de l'Anatomie et de la Physiologie. 1, 69-87 .</li><li>Bunster, E., Meyer, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+methods+of+parabiosis.">Improved methods of parabiosis.</a> Anat. Rec. 57, 339-380 (1933).</li><li>Waskow, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+parabiotic+mice+for+the+study+of+DC+and+DC+precursor+circulation.">Generation of parabiotic mice for the study of DC and DC precursor circulation.</a> Methods Mol. Biol. 595, 413-428 (2010).</li><li>Finerty, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parabiosis+in+physiological+studies.">Parabiosis in physiological studies.</a> Physiol. Rev. 32, 277-302 (1952).</li><li>Aicher, A., Heeschen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonbone+marrow-derived+endothelial+progenitor+cells:+what+is+their+exact+location.">Nonbone marrow-derived endothelial progenitor cells: what is their exact location.</a> Circ. Res. 101, e102 (2007).</li><li>Abe, S., Boyer, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cells+derived+from+the+circulation+contribute+to+the+repair+of+lung+injury.">Cells derived from the circulation contribute to the repair of lung injury.</a> Am. J. Respir. Crit. Care Med. 170, 1158-1163 (2004).</li><li>Donskoy, E., Goldschneider, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thymocytopoiesis+is+maintained+by+blood-borne+precursors+throughout+postnatal+life.+A+study+in+parabiotic+mice.">Thymocytopoiesis is maintained by blood-borne precursors throughout postnatal life. A study in parabiotic mice.</a> J. Immunol. 148, 1604-1612 (1992).</li><li>Powell, A. E., Anderson, E. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fusion+between+Intestinal+epithelial+cells+and+macrophages+in+a+cancer+context+results+in+nuclear+reprogramming.">Fusion between Intestinal epithelial cells and macrophages in a cancer context results in nuclear reprogramming.</a> Cancer Res. 71, 1497-1505 (2011).</li><li>Duyverman, A. M., Kohno, 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=A+transient+parabiosis+skin+transplantation+model+in+mice.">A transient parabiosis skin transplantation model in mice.</a> Nat. Protoc. 7, 763-770 (2012).</li><li>Ajami, B., Bennett, J. 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=Local+self-renewal+can+sustain+CNS+microglia+maintenance+and+function+throughout+adult+life.">Local self-renewal can sustain CNS microglia maintenance and function throughout adult life.</a> Nat. Neurosci. 10, 1538-1543 (2007).</li><li>Weissman, I. L., Jerabek, 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=Tolerance+and+the+H-Y+antigen:+Requirement+for+male+T+cells,+but+not+B+cells,+to+induce+tolerance+in+neonatal+female+mice.">Tolerance and the H-Y antigen: Requirement for male T cells, but not B cells, to induce tolerance in neonatal female mice.</a> Transplantation. 37, 3-6 (1984).</li></ol>

Los autores no tienen intereses financieros en competencia.

El método parabiosis discutido aquí presenta dificultades técnicas mínimas y los resultados de las tasas de mortalidad bajas. La unión de las articulaciones de rodilla y de codo es una mejora significativa de la Bunster y la técnica Meyer. Sin embargo, el procedimiento sigue siendo invasiva así el mantenimiento de las condiciones estériles durante toda la cirugía es imperativo. Para prevenir una infección del sitio quirúrgico, es importante que los animales parabiosed reciben una combinación de antibióticos...

Parabiosis, uniéndose a la cirugía de dos organismos, fue descrita por primera vez en 1864 por Paul Bert como una manera de desarrollar un modelo para estudiar los sistemas circulatorios compartidos y consistió en la unión de la piel y paredes musculares de dos ratas 1. Parabiosis promueve la formación de la microvasculatura en el sitio de la inflamación 3 y ha tenido varias aplicaciones en estudios fisiológicos, tales como la comunicación hormonal entre la glándula pituitaria y de las gónadas, así como la función del riñón en la hipertensión 4. Se ha empleado más para investigar la contratación y la integración de las células progenitoras de la neovascularización 5, la migración de las células madre hematopoyéticas 6, y el tráfico de linfocitos 7, así como el papel y la cinética de células inflamatorias circulantes y tallos en la metástasis tumoral 8,9, y enfermedad neurodegenerativa 10. Una ventaja importante de las mentiras parabiosisen que los animales asociado comparten antígenos circulantes comunes, lo que permite la migración celular y la neovascularización sin desencadenar una reacción inmunológica. Es importante destacar que, Weissman et al. Han demostrado que parabiosis entre los ratones macho y hembra no conduce a la formación de anticuerpos anti HY anticuerpos 11. En el protocolo original descrito por Paul Bert los dos animales se unen entre sí a través de la conexión de la piel y paredes musculares 1. Este método, sin embargo, causó una gran tensión a los animales y se tradujo en una alta mortalidad debido a la infección de la herida. Desde entonces la técnica parabiosis ha sido revisado por varios grupos con ser el más predominante el protocolo propuesto por Bunster y Meyer en 1933 2. Su método incluye la unión de las articulaciones de escápula, las cavidades del cuerpo, y la piel, lo que permite un mejor apoyo y menos dolor para los animales. Al mismo tiempo, el nuevo método dio como resultado en la atención y sig post-operatorio mínimodisminuyó significativamente las tasas de mortalidad. El protocolo descrito en el presente documento es una modificación de la técnica de Bunster y Meyer que es menos invasiva y permite más firme unión. A saber, los ratones se conectan a través de las articulaciones del codo y de la rodilla, así como la piel. Esto evita que unirse a la extensión de la piel y por lo tanto causa menos dolor y las complicaciones. Aquí se describe la unión de un tipo salvaje (WT) de ratón adulto a un GFP constitutiva expresando ratón. Mostramos que dos semanas después de la cirugía que pueden alcanzar 50% de quimerismo en la sangre que demuestra la eficacia de este procedimiento quirúrgico para crear un sistema circulatorio compartido.

<table border="1">
	<tbody>
		<tr>
			<td>Name of equipment</td>
			<td>Company</td>
			<td>Catalog number</td>
		</tr>
		<tr>
			<td>Isoflurane-Phoenix</td>
			<td>Clipper</td>
			<td>NDC 57319-559-06</td>
		</tr>
		<tr>
			<td>Posi-Seal Induction Chamber</td>
			<td>Molecular Imaging Products</td>
			<td>AS-01-0530-SM</td>
		</tr>
		<tr>
			<td>Portable Anesthesia System</td>
			<td>Molecular Imaging Products</td>
			<td>AS-01-0007</td>
		</tr>
		<tr>
			<td>Gaymer T Pump</td>
			<td>Gaymar Industries, Inc</td>
			<td>TP650</td>
		</tr>
		<tr>
			<td>Warming Blanket (Heating pad)</td>
			<td>Kent Scientific Corp</td>
			<td>TP-22G</td>
		</tr>
		<tr>
			<td>Curved forceps</td>
			<td>Roboz</td>
			<td>RS-5101</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Fine Science Tools (FST)</td>
			<td>FST 14063-09</td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>FST</td>
			<td>FST 12501-13</td>
		</tr>
		<tr>
			<td>Electrical shaver</td>
			<td>Oster</td>
			<td>Golden A5</td>
		</tr>
	</tbody>
</table>

parabiosis in mice: a detailed protocol

Parabiosis es una unión quirúrgica de dos organismos que permiten el intercambio de la circulación sanguínea. Colocación de la piel de dos animales promueve la formación de la microvasculatura en el sitio de la inflamación. Socios parabiotic comparten sus antígenos circulantes y por lo tanto están libres de la reacción inmunitaria adversa. Descrita por primera vez por Paul Bert en 1864 1, la cirugía parabiosis fue refinado por Bunster y Meyer en 1933 para mejorar la supervivencia de los animales 2. En el protocolo actual, dos ratones se unen quirúrgicamente tras una modificación de la técnica de Bunster y Meyer. Los animales están conectados a través de la articulación del codo y de la rodilla, seguido de la unión de la piel permitiendo un apoyo firme que impide que la tensión en la piel suturada. En este documento, se describe en detalle el parabiotic unión de un GFP ubicua expresando ratón a una de tipo salvaje (WT) de ratón. Dos semanas después del procedimiento, el par se separa y células positivas para GFP se puede detectar por análisis de citometría de flujo en la sangre circulanteion del ratón PESO. El quimerismo de la sangre permite examinar la contribución de las células circulantes de un animal en el otro.

El resultado anticipado de parabiosis de dos organismos es la contribución igual de sistema circulatorio de cada animal a una circulación de la sangre común (Figura 2). Se puede verificar fácilmente el equilibrado exitoso de la sangre de la parabiosed WT y ratones GFP positivos por análisis de citometría de flujo. Aquí, la sangre venosa se obtuvo de las colas de ambos parabionts a las 2 semanas después de la cirugía y se fraccionó para las células de sangre periférica (para excluir eritrocit...

Watch this Scientific Journal Video about Parabiosis in Mice: A Detailed Protocol at JoVE.com

Parabiosis in Mice: A Detailed Protocol

Parabiotic unión de dos organismos conduce al desarrollo de un sistema circulatorio compartido. En este protocolo, se describen los pasos quirúrgicos para formar una conexión parabiotic entre un ratón de tipo salvaje y un ratón que expresa GFP constitutiva.

Parabiosis en ratones: un protocolo detallado

Parabiosis is a surgical union of two organisms allowing sharing of the blood circulation. Attaching the skin of two animals promotes formation of ...

Research

JoVE Journal

Medicine

73.7K vistas.  University of California, Los Angeles.  Parabiotic unión de dos organismos conduce al desarrollo de un sistema circulatorio compartido. En este protocolo, se describen los pasos quirúrgicos para formar una conexión parabiotic entre un ratón de tipo salvaje y un ratón que expresa GFP constitutiva. 

Video: Parabiosis in Mice: A Detailed Protocol

The Trier Social Stress Test Protocol for Inducing Psychological Stress

This article demonstrates a psychological stress protocol for use in a laboratory setting. Protocols that allow researchers to study the biological pathways of the stress response in health and disease are fundamental to the progress of research in stress and anxiety.1 Although numerous protocols exist for inducing stress response in the laboratory, many neglect to provide a naturalistic context or to incorporate aspects of social and psychological stress. Of psychological stress protocols, meta-analysis suggests that the Trier Social Stress Test (TSST) is the most useful and appropriate standardized protocol for studies of stress hormone reactivity.2 In the original description of the TSST, researchers sought to design and evaluate a procedure capable of inducing a reliable stress response in the majority of healthy volunteers.3 These researchers found elevations in heart rate, blood pressure and several endocrine stress markers in response to the TSST (a psychological stressor) compared to a saline injection (a physical stressor).3 Although the TSST has been modified to meet the needs of various research groups, it generally consists of a waiting period upon arrival, anticipatory speech preparation, speech performance, and verbal arithmetic performance periods, followed by one or more recovery periods. The TSST requires participants to prepare and deliver a speech, and verbally respond to a challenging arithmetic problem in the presence of a socially evaluative audience.3 Social evaluation and uncontrollability have been identified as key components of stress induction by the TSST.4 In use for over a decade, the goal of the TSST is to systematically induce a stress response in order to measure differences in reactivity, anxiety and activation of the hypothalamic-pituitary-adrenal (HPA) or sympathetic-adrenal-medullary (SAM) axis during the task.1 Researchers generally assess changes in self-reported anxiety, physiological measures (e.g. heart rate), and/or neuroendocrine indices (e.g. the stress hormone cortisol) in response to the TSST. Many investigators have adopted salivary sampling for stress markers such as cortisol and alpha-amylase (a marker of autonomic nervous system activation) as an alternative to blood sampling to reduce the confounding stress of blood-collection techniques. In addition to changes experienced by an individual completing the TSST, researchers can compare changes between different treatment groups (e.g. clinical versus healthy control samples) or the effectiveness of stress-reducing interventions.1

This article describes a protocol for inducing psychological stress in participants, which enables researchers to measure psychological, physiological and neuroendocrine responses to stress within single participants or between groups.

El estrés social Trier Protocolo de prueba para la inducción de estrés psicológico

This article demonstrates a psychological stress protocol for use in a laboratory setting.  Protocols that allow researchers to study the biological ...

Investigación

Medicina

Protocol for Long Duration Whole Body Hyperthermia in Mice

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body temperature that is observed during fever. The use of hyperthermia as an adjuvant has emerged as a promising procedure for tumor regression in the field of cancer biology. For this purpose, the most important requirement is to have reliable and uniform heating protocols. We have developed a protocol for hyperthermia (whole body) in mice. In this protocol, animals are exposed to cycles of hyperthermia for 90 min followed by a rest period of 15 min. During this period mice have easy access to food and water. High body temperature spikes in the mice during first few hyperthermia exposure cycles are prevented by immobilizing the animal. Additionally, normal saline is administered in first few cycles to minimize the effects of dehydration. This protocol can simulate fever like conditions in mice up to 12-24 hr. We have used 8-12 weeks old BALB/Cj female mice to demonstrate the protocol.

This paper describes a protocol for whole body hyperthermia in mice that can stimulate fever like conditions up to 12-24 hr.

Protocolo para la hipertermia corporal Larga Duración Total en ratones

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body ...

Collection Protocol for Human Pancreas

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation of pancreas recovered from cadaveric organ donors. The pancreas is divided into 3 main regions (head, body, tail) followed by serial transverse sections throughout the medial to lateral axis. Alternating sections are used for fixed paraffin and fresh frozen blocks and remnant samples are minced for snap frozen sample preparations, either with or without RNAse inhibitors, for DNA, RNA, or protein isolation. The overall goal of the pancreas dissection procedure is to sample the entire pancreas while maintaining anatomical orientation.
Endocrine cell heterogeneity in terms of islet composition, size, and numbers is reported for human islets compared to rodent islets 1. The majority of human islets from the pancreas head, body and tail regions are composed of insulin-containing &beta;-cells followed by lower proportions of glucagon-containing &alpha;-cells and somatostatin-containing &delta;-cells. Pancreatic polypeptide-containing PP cells and ghrelin-containing epsilon cells are also present but in small numbers. In contrast, the uncinate region contains islets that are primarily composed of pancreatic polypeptide-containing PP cells 2. These regional islet variations arise from developmental differences. The pancreas develops from the ventral and dorsal pancreatic buds in the foregut and after rotation of the stomach and duodenum, the ventral lobe moves and fuses with the dorsal 3. The ventral lobe forms the posterior portion of the head including the uncinate process while the dorsal lobe gives rise to the rest of the organ. Regional pancreatic variation is also reported with the tail region having higher islet density compared to other regions and the dorsal lobe-derived components undergoing selective atrophy in type 1 diabetes4,5.
Additional organs and tissues are often recovered from the organ donors and include pancreatic lymph nodes, spleen and non-pancreatic lymph nodes. These samples are recovered with similar formats as for the pancreas with the addition of isolation of cryopreserved cells. When the proximal duodenum is included with the pancreas, duodenal mucosa may be collected for paraffin and frozen blocks and minced snap frozen preparations.

This video demonstrates a dissection procedure for processing human pancreas into multiple storage formats. Anatomical orientation is maintained throughout the pancreatic regions to allow definition of regional islet composition and density.

Colección Protocolo de páncreas humano

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation ...

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

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

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

Infusiones de la arteria carótida para farmacocinéticas y farmacodinámicas Análisis de taxanos en ratones

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

A Simple Critical-sized Femoral Defect Model in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility. 
Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue.
Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects in mice.

A-crítico tamaño femoral Defecto sencillo modelo en ratones

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

Heterotopic Renal Autotransplantation in a Porcine Model: A Step-by-Step Protocol

Kidney transplantation is the treatment of choice for patients suffering from end-stage renal disease. It offers better life expectancy and higher quality of life when compared to dialysis. Although the last few decades have seen major improvements in patient outcomes following kidney transplantation, the increasing shortage of available organs represents a severe problem worldwide. To expand the donor pool, marginal kidney grafts recovered from extended criteria donors (ECD) or donated after circulatory death (DCD) are now accepted for transplantation. To further improve the postoperative outcome of these marginal grafts, research must focus on new therapeutic approaches such as alternative preservation techniques, immunomodulation, gene transfer, and stem cell administration.
Experimental studies in animal models are the final step before newly developed techniques can be translated into clinical practice. Porcine kidney transplantation is an excellent model of human transplantation and allows investigation of novel approaches. The major advantage of the porcine model is its anatomical and physiological similarity to the human body, which facilitates the rapid translation of new findings to clinical trials. This article offers a surgical step-by-step protocol for an autotransplantation model and highlights key factors to ensure experimental success. Adequate pre- and postoperative housing, attentive anesthesia, and consistent surgical techniques result in favorable postoperative outcomes. Resection of the contralateral native kidney provides the opportunity to assess post-transplant graft function. The placement of venous and urinary catheters and the use of metabolic cages allow further detailed evaluation. For long-term follow-up studies and investigation of alternative graft preservation techniques, autotransplantation models are superior to allotransplantation models, as they avoid the confounding bias posed by rejection and immunosuppressive medication.

Porcine models of organ transplantation provide an important platform to study mechanisms of organ preservation. This article describes a heterotopic porcine renal autotransplantation model, which allows investigating new approaches to improve the outcome of transplantation using marginal kidney grafts.

Heterotópico renal autotrasplante en un modelo porcino: Un Protocolo de Paso a Paso

Kidney transplantation is the treatment of choice for patients suffering from end-stage renal disease. It offers better life expectancy and higher quality ...

A Step by Step Protocol for Subretinal Surgery in Rabbits

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in adults in industrially developed countries. RPE transplantation appears to be a promising therapy, as it may replace dysfunctional RPE, restore its function, and thereby vision.
Here we describe a method for transplanting a cultured RPE monolayer on a scaffold into the subretinal space (SRS) of rabbits. After vitrectomy xenotransplants were delivered into the SRS using a custom made shooter consisting of a 20-gauge metallic nozzle with a polytetrafluoroethylene (PTFE) coated plunger. The current technique evolved in over 150 rabbit surgeries over 6 years. Post-operative follow-up can be obtained using non-invasive and repetitive in vivo imaging such as spectral domain optical coherence tomography (SD-OCT) followed by perfusion-fixed histology.
The method has well-defined steps for easy learning and high success rate. Rabbits are considered a large eye animal model useful in preclinical studies for clinical translation. In this context rabbits are a cost-efficient and perhaps convenient alternative to other large eye animal models.

Retinal pigment epithelium (RPE) replacement strategies and gene-based therapy are considered for several retinal degenerative conditions. For clinical translation, large eye animal models are required to study surgical techniques applicable in patients. Here we present a rabbit model for subretinal surgery geared towards RPE transplantation, which is versatile and cost-efficient.

Un Paso a Paso Protocolo para la cirugía subretiniana en conejos

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in ...

Creation of Abdominal Adhesions in Mice

Abdominal adhesions consist of fibrotic tissue that forms in the peritoneal space in response to an inflammatory insult, typically surgery or intraabdominal infection. The precise mechanisms underlying adhesion formation are poorly understood. Many compounds and physical barriers have been tested for their ability to prevent adhesions after surgery with varying levels of success. The mouse and rat are important models for the study of abdominal adhesions. Several different techniques for the creation of adhesions in the mouse and rat exist in the literature. Here we describe a protocol utilizing abrasion of the cecum with sandpaper and sutures placed in the right abdominal sidewall. The mouse is anesthetized and the abdomen is prepped. A midline laparotomy is created and the cecum is identified. Sandpaper is used to gently abrade the surface of the cecum. Next, several figure-of-eight sutures are placed into the peritoneum of the right abdominal sidewall. The abdominal cavity is irrigated, a small amount of starch is applied, and the incision is closed. We have found that this technique produces the most consistent adhesions with the lowest mortality rate.

Abdominal adhesions that form after surgery are a major cause of pain, infertility, and hospitalization and reoperation for small bowel obstruction. Our surgical procedure for creating abdominal adhesions in mice is a reliable tool to study the mechanisms underlying the formation of adhesions.

Creación de abdominales adherencias en ratones

Abdominal adhesions consist of fibrotic tissue that forms in the peritoneal space in response to an inflammatory insult, typically surgery or ...

A Detailed Protocol for Perspiration Monitoring Using a Novel, Small, Wireless Device

Perspiration monitoring can be utilized for the detection of certain diseases, such as thermoregulation and mental disorders, particularly when the patients are unaware of such disorders or are having difficulty expressing their symptoms. Until now, several devices for perspiration monitoring have been developed; however, such devices tend to have a relatively large exterior, considerable power consumption, and/or less sensitivity.
Recently, we developed a small, wireless device for perspiration monitoring. The device consists of a temperature/relative humidity (T/RH) sensor, battery-driven small data logger, and silica gel as a desiccant in a small cylindrical exterior. The T/RH sensor is placed between the detection windows (through which the water vapor from the skin enters) and the silica gel. The underlying principle of the perspiration monitoring device is based on Fick&#39;s law of diffusion, which means that water vapor flux from the skin to the silica gel (i.e.&#160;transepidermal water loss and perspiration) can be captured by change in humidity at the T/RH sensor. In addition, a baseline subtraction method was adopted to distinguish perspiration and transepidermal water loss.
As shown in the previous report, the developed device can monitor the perspiration at any sites of the body in an easy, wireless manner. However, detailed methods of how to use the device have not been disclosed yet. In this article, therefore, we would like to show the point-by-point tutorials of how to use the device for perspiration monitoring, by showing the sympathetic activity test with the sympathetic skin response monitoring as an example.

Recently, we developed a small wireless device for perspiration monitoring. In this article, we present detailed protocols on how to use the device for perspiration monitoring with an example of the sympathetic activity test.

Un protocolo detallado para la Vigilancia transpiración utilizando un nuevo, pequeño, dispositivo inalámbrico

Perspiration monitoring can be utilized for the detection of certain diseases, such as thermoregulation and mental disorders, particularly when the ...

A Detailed Protocol for Physiological Parameters Acquisition and Analysis in Neurosurgical Critical Patients

Intracranial pressure (ICP) monitoring is now widely used in neurosurgical critical patients. Besides mean ICP value, the ICP derived parameters such as ICP waveform, amplitude of pulse (AMP), the correlation of ICP amplitude and ICP mean (RAP), pressure reactivity index (PRx), ICP and arterial blood pressure (ABP) wave amplitude correlation (IAAC), and so on, can reflect intracranial status, predict prognosis, and can also be used as guidance of proper treatment. However, most of the clinicians focus only on the mean ICP value while ignoring these parameters because of the limitations of the current devices. We have recently developed a multimodality monitoring system to address these drawbacks. This portable, user-friendly system will use a data collecting and storing device to continuously acquire patients&#39; physiological parameters first, i.e., ABP, ICP, and oxygen saturation, and then analyze these physiological parameters. We hope that the multimodality monitoring system will be accepted as a key measure to monitor physiological parameters, to analyze the current clinical status, and to predict the prognosis of the neurosurgical critical patients.

Recently, we have developed a portable multimodality monitoring system for the monitoring of various physiological parameters in neurosurgical critical patients. Detailed protocols on how to use this multimodality monitoring system are presented here.

Un protocolo detallado de parámetros fisiológicos de adquisición y análisis en pacientes críticos neuroquirúrgicos

Intracranial pressure (ICP) monitoring is now widely used in neurosurgical critical patients. Besides mean ICP value, the ICP derived parameters such as ...