The authors have no acknowledgements.

NOTA: Todos los animales procedimientos fueron realizados con la aprobación del Comité de Cuidado y Uso de Animales Institucional (IACUC) de la Universidad de Cincinnati y de acuerdo con la Guía para el Cuidado y Uso de Animales de Laboratorio de los Institutos Nacionales de Salud (NIH Publication No . 85-23, revisado 1996). 1. La eutanasia y esterilizar el ratón <ol><li> Coloca el ratón en un dropbox que contiene una dosis supratheraputic de isoflurano y permitir a inhalar a efecto. Una vez que el ratón es sacrificado, retirar de la caja. </li><li> Cervical dislocar como un segundo medio de la eutanasia. </li><li> Esterilizar la superficie externa del ratón por la limpieza del animal con etanol al 70%. </li></ol> 2. Identificación y aislamiento de tres diferentes depósitos adiposos <ol><li> Tejido adiposo marrón (BAT) Aislamiento: <ol><li> Asegúrese de que la piel está húmeda de la limpieza de alcohol para ayudar a la corte a través de la epidermis y la dermis layers. </li><li> Coloque el ratón en una posición supina con la espalda contra la mesa. </li><li> Coge la piel justo debajo del diafragma con fórceps, ascensor y una incisión con tijeras. </li><li> Cortar transversalmente alrededor de la circunferencia del ratón para exponer el peritoneo. </li><li> Revelar la BAT depósito en forma de mariposa por degloving la mitad superior del ratón. Mantenga los apéndices inferiores y el abdomen en una mano y tirando de la piel hacia la cabeza. </li><li> Orientar el ratón, de modo que se coloca boca abajo en la mesa. Tenga cuidado de no contaminar el depósito expuesto con el pelo. </li><li> Instrumentos quirúrgicos limpios y el cambio a un nuevo par de guantes. </li><li> Busque la escápula y depósito correspondiente. Retire con cuidado el adiposo blanco superficial encima de la mariposa y luego diseccionar la mariposa de la grasa marrón interescapular. Tenga cuidado de evitar que el músculo estrechamente asociado con la grasa parda. NOTA: El uso de un microscopio de disección se recomienda para la eliminación de la white adiposo, así como en la separación de la adiposo marrón de las escápulas. </li><li> Quite la grasa de depósito y transferir a un tubo de microcentrífuga de 2 ml. </li><li> Si el ARN o proteína es a extraer, congelar el tejido por inmersión en nitrógeno líquido y se almacena a -80 ° C. Congelar la muestra a la vez para evitar la degradación. Si el cultivo, cubrir el tejido en DMEF-12 y establecer en hielo hasta que se recogen todas las muestras para cultivo (información complementaria). </li></ol></li><li> Aislamiento de tejido adiposo subcutáneo (SC), un blanco tejido adiposo Depot (WAT): <ol><li> Póngase un par de guantes nuevos como a la contaminación cruzada entre los depósitos de grasa. </li><li> Revelar la inguinal, depósitos SQ triangulares por DE-enguantado la mitad inferior del ratón. Mantenga los apéndices superiores y tórax en una mano y tirando de la piel hacia abajo hacia los pies con la otra mano. </li><li> Oriente el ratón en una posición supina con cuidado de no contaminar el depósito expuesto con el pelo. </li><li> Surgica Cleaninstrumentos l y el cambio a un nuevo par de guantes. </li><li> Diseccionar cuidadosamente los triángulos de grasa SQ. Tenga cuidado de no contaminar la muestra con el músculo, la vecina de grasa, glándulas mamarias o cortando cualquier barco y contaminar la muestra de sangre. NOTA: El uso de un microscopio de disección se recomienda si las fronteras no están claramente definidos. </li><li> Retire los depósitos de grasa y la transferencia a 2 ml tubos de microcentrífuga. </li><li> Si el ARN o proteína es a extraer, congelar el tejido por inmersión en nitrógeno líquido y se almacena a -80 ° C. Congelar la muestra a la vez para evitar la degradación. </li><li> Para la tinción, arreglar o incrustar en octubre Si el cultivo, cubrir el tejido en DMEF-12 y establecer en hielo hasta que se recogen todas las muestras para cultivo (información complementaria). </li></ol></li><li> Aislamiento de gonadal Fat un tejido adiposo visceral (IVA) y el tejido adiposo blanco (WAT) Depot: <ol><li> Póngase un par de guantes nuevos como a la contaminación cruzada entre los depósitos de grasa. </li><li> Con cienciassors, cortar el peritoneo transversalmente, directamente debajo del diafragma. Cortar el peritoneo desde el diafragma hasta mediados recto coronalmente para exponer los órganos abdominales. </li><li> Localizar los testículos o los ovarios e identificar el tejido adiposo blanco adjunto, conocido como el adiposo del epidídimo en varones o adiposo gonadal en las hembras. </li><li> Instrumentos quirúrgicos limpios y el cambio a un nuevo par de guantes </li><li> Diseccionar cuidadosamente ambos depósitos de grasa del epidídimo de los testículos, epidídimos y conductos deferentes. O si es mujer, diseccionar cuidadosamente ambas almohadillas de grasa gonadal de los ovarios. </li><li> Retire los depósitos de grasa y transferirlos a 2 tubos de microcentrífuga ml. </li><li> Si el ARN o proteína es a extraer, congelar el tejido por inmersión en nitrógeno líquido y se almacena a -80 ° C. Congelar la muestra a la vez para evitar la degradación. </li><li> Para la tinción, arreglar o incrustar en octubre Si el cultivo, cubrir el tejido en DMEF-12 y establecer en hielo hasta que se recogen todas las muestras para cultivo (información complementaria). </li&gt; </ol></li></ol> 3. Aislamiento de tejido adiposo perivascular (IVAP) <ol><li> Aislamiento del Corazón: <ol><li> Coloque el ratón en una posición supina con apéndices superior e inferior extendidas hacia afuera. </li><li> Apéndices Seguros que utilizan una cinta quirúrgica. </li><li> Después de colocar el ratón como se indica anteriormente, crear tensión levantando el proceso xifoides con fórceps. Cortar horizontalmente a través del diafragma, la exposición de la parte inferior de la cavidad torácica. </li><li> Mientras mantiene la tensión, levantando el proceso xifoides, cortar a través de la caja torácica superiormente hacia la cabeza, sólo para el lado del esternón. </li><li> Recoger la caja torácica justo por debajo de la clavícula, y cortar a lo largo de la longitud inferior de la clavícula hacia fuera hacia la axila en ambas direcciones. La cavidad torácica y su contenido (corazón, pulmones, etc.) deben ahora ser claramente visible. </li><li> Limpie la cavidad torácica de la sangre ajena y líquido utilizando ga estériluze para absorber el fluido. Si órganos o recipientes colectores asegúrese de profuso (información complementaria). </li><li> Una vez que el área se borra de líquido, cortar los vasos y bronquios de fijación para eliminar los pulmones, lo que permite una mejor exposición del corazón. </li></ol></li><li> Localización y Escisión de tejido adiposo perivascular aórtica (IVAP): <ol><li> Quite los siguientes órganos como para identificar mejor las porciones inferiores de la aorta: hígado, estómago, bazo, páncreas, intestinos y colon. </li><li> En primer lugar comenzar con la identificación del estómago y el esófago. Cortar el esófago en la unión gastro-esofágica para liberar el estómago del cuerpo. </li><li> A continuación, identificar los intestinos y el mesenterio circundante. Cortar superficialmente a través de mesenterio, ya que se encuentra muy de cerca a la porción renal de la aorta, luego &quot;ejecutar el intestino.&quot; </li><li> Cortar el colon libre como cerca del recto posible. Por lo tanto, liberando el estómago, intestino y colon de ratón. </li><li> Remover el estómago, intestino, colon, páncreas y el bazo cortando a través del mesenterio de fijación y los vasos. El páncreas y el bazo deben venir libre con el estómago, intestino y colon. </li><li> Retire el hígado por el corte a través de las venas hepáticas y adjuntando mesenterio, retire todos los lóbulos. </li><li> Cortar la capa visceral y la grasa rodean los riñones. Deja los riñones unidos a la aorta in vivo para servir como marcadores geográficos para los diferentes segmentos de la aorta. </li><li> Enjuague el área con 1x PBS estéril y eliminar todo el fluido por absorción con una gasa estéril. </li><li> El uso de micro-tijeras y micro-pinzas, separar la aorta de su apego a la espina dorsal y su enganche ventral al esófago. </li><li> Aislar la aorta siguiendo y desconexión de la aorta de la longitud de la aorta descendente desde el origen en el corazón hasta la bifurcación en la región ilíaca. </li><li> Identificar y aislar los vasos subclavia. Aislar estas embarcacionesdesde el cuello hasta la raíz aórtica para exponer mejor la unión aórtica y la raíz en el corazón. </li><li> Retire el timo a continuación, cortar el tronco braquiocefálico, la arteria carótida común izquierda y la arteria subclavia izquierda, permitiendo el movimiento del corazón. </li><li> Con la ayuda de un microscopio de disección, ver la capa de tejido adiposo perivascular (IVAP) que rodea la aorta. </li><li> Teniendo mucho cuidado para no pellizcar o apretar el IVAP, tire suavemente de la manera IVAP de la aorta con micro-forceps. Cortar suavemente la fijación de la IVAP a la aorta con micro-tijeras a partir de la región torácica justo por encima de donde se encuentra el diafragma. NOTA: Se recomienda utilizar un microscopio de disección. </li><li> Repita este proceso toda la longitud de la aorta, terminando en la región de la aorta infrarrenal, que se encuentra justo por encima de la bifurcación ilíaca del vaso aórtico. </li><li> Si se desea arco IVAP aórtica, que se utiliza el mismo método para eliminar el IVAP de la menor curvature del arco. </li><li> Coloque las muestras IVAP en 2 ml tubo (s) de microcentrífuga. </li><li> Si el ARN o proteína es a extraer, congelar el tejido por inmersión en nitrógeno líquido y se almacena a -80 ° C. Congelar la muestra a la vez para evitar la degradación. Si el cultivo, cubrir el tejido en DMEF-12 y establecer en hielo hasta que se recogen todas las muestras para cultivo (información complementaria). NOTA: los depósitos adiposos adicionales a tener en cuenta si está interesado en un análisis exhaustivo de depósito adiposo incluye: retroperitoneal, mesentérica, omental, pericárdico y poplítea. </li></ol></li></ol>

<ol>
	<li>Ogden, C. L., Carroll, M. D., Kit, B. K., Flegal, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+of+obesity+in+the+United+States,+2009-2010.">Prevalence of obesity in the United States, 2009-2010.</a> NCHS Data Brief. 82, 1-8 (2012).</li><li>Flegal, K. M., Carroll, M. D., Kit, B. K., Ogden, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+of+obesity+and+trends+in+the+distribution+of+body+mass+index+among+US+adults,+1999-2010.">Prevalence of obesity and trends in the distribution of body mass index among US adults, 1999-2010.</a> 307 (5), 491-497 (2012).</li><li>Cawley, J., Meyerhoefer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+medical+care+costs+of+obesity:+an+instrumental+variables+approach.">The medical care costs of obesity: an instrumental variables approach.</a> Journal of Health Economics. 31 (1), 219-230 (2012).</li><li>Marder, W., Chang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Childhood+Obesity:+Costs,+Treatment+Patterns,+Disparities+in+Care,+and+Prevalent+Medical+Conditions.">Childhood Obesity: Costs, Treatment Patterns, Disparities in Care, and Prevalent Medical Conditions.</a> Thomson Medstat Research Brief. , (2006).</li><li>Cortez, M., Carmo, L. S., Rogero, M. M., Borelli, P., Fock, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-fat+diet+increases+IL-1,+IL-6,+and+TNF-&#945;+production+by+increasing+NF-&#954;B+and+attenuating+PPAR-&#947;+expression+in+bone+marrow+mesenchymal+stem+cells.">A high-fat diet increases IL-1, IL-6, and TNF-&#945; production by increasing NF-&#954;B and attenuating PPAR-&#947; expression in bone marrow mesenchymal stem cells.</a> Inflammation. 36 (2), 379-386 (2013).</li><li>Sanchez-Gurmaches, J., Hung, C. M., Sparks, C. A., Tang, Y., Li, H., Guertin, 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=PTEN+loss+in+the+Myf5+lineage+redistributes+body+fat+and+reveals+subsets+of+white+adipocytes+that+arise+from+Myf5+precursors.">PTEN loss in the Myf5 lineage redistributes body fat and reveals subsets of white adipocytes that arise from Myf5 precursors.</a> Cell Metab. 16 (3), 348-362 (2012).</li><li>Sethi, J. K., Vidal-Puig, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thematic+review+series:+adipocyte+biology.+Adipose+tissue+function+and+plasticity+orchestrate+nutritional+adaptation.">Thematic review series: adipocyte biology. Adipose tissue function and plasticity orchestrate nutritional adaptation.</a> J Lipid Res. 48 (6), 1253-1262 (2007).</li><li>Aarsland, A., Chinkes, D., Wolfe, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+and+whole-body+fat+synthesis+in+humans+during+carbohydrate+overfeeding.">Hepatic and whole-body fat synthesis in humans during carbohydrate overfeeding.</a> Am J Clin Nutr. 65 (6), 1774-1782 (1997).</li><li>Park, A., Kim, W. K., Bae, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinction+of+white,+beige+and+brown+adipocytes+derived+from+mesenchymal+stem+cells.">Distinction of white, beige and brown adipocytes derived from mesenchymal stem cells.</a> World J. Stem Cells. 6 (1), 33-42 (2014).</li><li>Wu, J., 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=Beige+adipocytes+are+a+distinct+type+of+thermogenic+fat+cell+in+mouse+and.">Beige adipocytes are a distinct type of thermogenic fat cell in mouse and.</a> 150 (2), 366-376 (2012).</li><li>Giralt, M., Villarroya, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=White,+brown,+beige/brite:+different+adipose+cells+for+different+functions.">White, brown, beige/brite: different adipose cells for different functions.</a> Endocrinology. 154 (9), 2992-3000 (2013).</li><li>Casteilla, L., P&#233;nicaud, L., Cousin, B., Calise, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choosing+an+adipose+tissue+depot+for+sampling:+factors+in+selection+and+depot+specificity.">Choosing an adipose tissue depot for sampling: factors in selection and depot specificity.</a> Methods Mol. Biol. 456, 23-38 (2008).</li><li>Grant, R., Youm, Y. H., Ravussin, A., Dixit, V. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+adipose+tissue+leukocytosis+in+obesity.">Quantification of adipose tissue leukocytosis in obesity.</a> Methods Mol. Biol. 1040, 195-209 (2013).</li></ol>

The authors declare that they have no competing financial interests or other conflicts of interest.

La obesidad puede conducir a una gran cantidad de morbilidades y la plena comprensión del papel que juega adiposo no se entiende completamente; Por lo tanto, la investigación continuada en el campo de la adiposo es necesario. Los modelos animales, específicamente modelos murinos son ideales para la investigación inicial en la progresión de las enfermedades y las pruebas de los posibles tratamientos farmacéuticos. En el uso de estos modelos, el aislamiento y la escisión precisa de los depósitos de tejido adiposo ...

Adiposo hizo una aparición notable en la atención de los medios, debido al aumento espectacular de la obesidad durante las últimas décadas del siglo 20. La obesidad afecta actualmente a más de un tercio de los adultos y el 17% de los niños y adolescentes en los Estados Unidos (US) 1. Que abarca todos los grupos étnicos, la investigación estadística que rodea la epidemia de la obesidad se ha demostrado que los negros no hispanos tienen la tasa ajustada por edad más alta de obesidad (49,5%) en comparación con los mexicano-americanos (40,4%), los hispanos (39.1%), y no los blancos no hispanos (34,3%) 2. El efecto económico de la obesidad es también una preocupación cada vez mayor para el sistema sanitario. En 2012, se estimó que el costo anual de la atención médica para la obesidad en los EE.UU. en 2005 fue de $ 190,2 mil millones, casi el 21% del presupuesto total de los gastos médicos. Tristemente, la obesidad infantil se estima que es responsable por $ 14 mil millones en costos médicos directos solo. Estadísticamente, se determinó que el costo médico promedio deindividuos con obesidad fue $ 2,741 superior al año que aquellos sin esta morbilidad 3-5. La obesidad es un factor de riesgo importante para una variedad de condiciones tales como: la diabetes tipo 2, dislipidemia, enfermedad cardiovascular, cáncer, trastornos esqueléticos musculares y la inflamación crónica. La obesidad está profundamente ligada a la patogénesis del síndrome metabólico y otras enfermedades crónicas 6-8. Con este tipo de conexiones drásticas y negativas entre la obesidad y otras comorbilidades, la investigación científica ha centrado la atención para comprender mejor la epidemia actual y los papeles diversos y esencial desempeñada por adiposo. Históricamente, el tejido adiposo se considera intrascendente y fue visto simplemente como un tejido simple relleno. Actualmente, adiposo se ha demostrado que desempeñan muchas funciones esenciales en la función del cuerpo en: metabolismo, la regulación hormonal, la inflamación, la protección y el aislamiento 9. El tejido adiposo está compuesto principalmente deadipocitos, pero también contiene los pericitos, células endoteliales, monocitos, macrófagos y células madre pluripotentes 8. El tejido adiposo se distribuye por todo el cuerpo en depósitos distintos. Los principales depósitos se pueden encontrar por vía subdérmica, subcutánea, intramuscular, y visceral 10. Depósitos adiposos se han demostrado tener unos perfiles metabólicos específicos de depósito, que han demostrado una susceptibilidad específica de depósito a la obesidad y trastornos relacionados 8. Tradicionalmente, el tejido adiposo se ha clasificado en dos tipos principales: tejido adiposo blanco (WAT) y tejido adiposo marrón (BAT); aunque la literatura reciente indica las presencias de un tercer grupo bautizaron adiposo brite o beige 11. El tejido adiposo se ha demostrado que tienen diferentes colores, morfologías, funciones metabólicas, las características bioquímicas y patrones de expresión genéticos 10. Los adipocitos en WAT tienen un único y gran gotas de lípidos y cantidades variables de las mitocondrias. WATse encuentra predominantemente en las localidades subcutáneos y viscerales del cuerpo. Funciones WAT principalmente como un sitio de almacenamiento de energía y la protección de órganos. Los adipocitos en BAT tienen una morfología multilocular y abundantes mitocondrias. BAT se encuentra principalmente en el cuello y los grandes vasos sanguíneos del tórax, así como las escápulas 12. BAT funciona principalmente en los comportamientos de gastar energía que regulan la termogénesis 7. Adiposo Brite o beige se ha demostrado que compartir una morfología y la expresión análoga a BAT pero se ha encontrado para ser originado a partir de adipocitos blancos 11. El método quirúrgico se describe en este manuscrito proporciona a los investigadores la capacidad de analizar los diferentes efectos que factores tales como: medio ambiente, productos farmacéuticos, y la genética, tienen sobre adiposo; así como el papel adiposo beneficioso o perjudicial juega en la enfermedad y la salud en general. Además, proporcionando una manera de identificar y aislar diferentes tipos de tejido adiposo parapermitirá comprender mejor las relaciones bioquímicas y diferencias entre los depósitos. Esto puede ayudar en la determinación de la relación entre la ubicación, la función y tipos de grasa en el cuerpo. Este método descrito logra esto proporcionando los medios para la visualización bruto, el análisis de la expresión génica, análisis de la expresión de proteínas, el examen histológico, y el aislamiento de líneas celulares primarias para estudios in vitro. Actualmente hay muchos artículos que proporcionan información sobre el comportamiento metabólico de los diferentes depósitos de tejido adiposo, así como sus localizaciones anatómicas; pero no proporcionan un método en profundidad sobre cómo localizar de forma específica, identificar y aislar a estos depósitos. Este método proporciona una técnica quirúrgica precisa que permite el aislamiento de múltiples depósitos con una cantidad mínima de la disección y la contaminación en comparación con otros métodos diseñados para el aislamiento de uno o dos depósitos 13-14. El objetivo de este protocolo es proporcionarun método preciso para la identificación y el aislamiento de diferentes tipos de depósitos de grasa desde múltiples localizaciones anatómicas.

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		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Name of Reagent/ Equipment</td>
			<td style="width:175px;">Company</td>
			<td style="width:125px;">Catalog Number</td>
			<td style="width:188px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">isoflurane</td>
			<td>Med-Vet International&#160;</td>
			<td>#RXISO-250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">70% Ethanol</td>
			<td style="width:175px;">Fisher</td>
			<td style="width:125px;">07-678-001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">DMEF-12</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:125px;">D-6421</td>
			<td>Warm in waterbath before putting on tissue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">2 mL microcentrifuge tubes</td>
			<td style="width:175px;">Midsci</td>
			<td style="width:125px;">MCT-200-C-S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Phosphate buffered saline</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:125px;">P5368-10PAK</td>
			<td></td>
		</tr>
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</table>

localization, identification, and excision of murine adipose depots

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 reached a staggering sum of $190.2 billion in direct medical costs alone. Obesity is a major risk factor for a wide host of diseases. Historically, little was known regarding adipose and its major and essential functions in the body. Brown and white adipose are the two main types of adipose but current literature has identified a new type of fat called brite or beige adipose. Research has shown that adipose depots have specific metabolic profiles and certain depots allow for a propensity for obesity and other related disorders. The goal of this protocol is to provide researchers the capacity to identify and excise adipose depots that will allow for the analysis of different factorial effects on adipose; as well as the beneficial or detrimental role adipose plays in disease and overall health. Isolation and excision of adipose depots allows investigators to look at gross morphological changes as well as histological changes. The adipose isolated can also be used for molecular studies to evaluate transcriptional and translational change or for in vitro experimentation to discover targets of interest and mechanisms of action. This technique is superior to other published techniques due to the design allowing for isolation of multiple depots with simplicity and minimal contamination.

Identificación y localización de inguinal adiposo subcutáneo, tejido adiposo marrón interescapular, adiposo del epidídimo visceral (Figura 1), así como el adiposo perivascular arco aórtico, la aorta torácica adiposo, tejido adiposo aórtica suprarrenal y el tejido adiposo de la aorta infrarrenal (Figura 2) se logró con éxito utilizando el método quirúrgico descrito. El examen histológico y la diferenciación entre muestras MTD y WAT fueron evaluados positivamente mediante h...

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Localization, Identification, and Excision of Murine Adipose Depots

Debido a la conexión drástico y negativo entre la obesidad y otras comorbilidades, la investigación sobre el papel adiposo desempeña en la enfermedad y la salud en general se justifica. Se presenta un protocolo para el aislamiento y la escisión de los depósitos de tejido adiposo que permitan el estudio de tejido adiposo usando in situ y métodos in vitro.

Localización, identificación y Escisión de depósitos adiposos murino

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 ...

localization-identification-and-excision-of-murine-adipose-depots

Research

JoVE Journal

Medicine

40.8K vistas.  University of Cincinnati College of Medicine.  Debido a la conexión drástico y negativo entre la obesidad y otras comorbilidades, la investigación sobre el papel adiposo desempeña en la enfermedad y la salud en general se justifica. Se presenta un protocolo para el aislamiento y la escisión de los depósitos de tejido adiposo que permitan el estudio de tejido adiposo usando in situ y métodos in vitro. 

Video: Localization, Identification, and Excision of Murine Adipose Depots

A Simplified Technique for In situ Excision of Cornea and Evisceration of Retinal Tissue from Human Ocular Globe

Enucleation is the process of retrieving the ocular globe from a cadaveric donor leaving the rest of the globe undisturbed. Excision refers to the retrieval of ocular tissues, especially cornea, by cutting it separate from the ocular globe. Evisceration is the process of removing the internal organs referred here as retina. The ocular globe consists of the cornea, the sclera, the vitreous body, the lens, the iris, the retina, the choroid, muscles etc (Suppl. Figure 1). When a patient is suffering from corneal damage, the cornea needs to be removed and a healthy one must be transplanted by keratoplastic surgeries. Genetic disorders or defects in retinal function can compromise vision. Human ocular globes can be used for various surgical procedures such as eye banking, transplantation of human cornea or sclera and research on ocular tissues. However, there is little information available on human corneal and retinal excision, probably due to the limited accessibility to human tissues. Most of the studies describing similar procedures are performed on animal models. Research scientists rely on the availability of properly dissected and well-conserved ocular tissues in order to extend the knowledge on human eye development, homeostasis and function. As we receive high amount of ocular globes out of which approximately 40% (Table 1) of them are used for research purposes, we are able to perform huge amount of experiments on these tissues, defining techniques to excise and preserve them regularly.
The cornea is an avascular tissue which enables the transmission of light onto the retina and for this purpose should always maintain a good degree of transparency. Within the cornea, the limbus region, which is a reservoir of the stem cells, helps the reconstruction of epithelial cells and restricts the overgrowth of the conjunctiva maintaining corneal transparency and clarity. The size and thickness of the cornea are critical for clear vision, as changes in either of them could lead to distracted, unclear vision. The cornea comprises of 5 layers; a) epithelium, b) Bowman's layer, c) stroma, d) Descemet's membrane and e) endothelium. All layers should function properly to ensure clear vision4,5,6. The choroid is the intermediate tunic between the sclera and retina, bounded on the interior by the Bruch's membrane and is responsible for blood flow in the eye. The choroid also helps to regulate the temperature and supplies nourishment to the outer layers of the retina5,6. The retina is a layer of nervous tissue that covers the back of the ocular globe (Suppl. Figure 1) and consists of two parts: a photoreceptive part and a non-receptive part. The retina helps to receive the light from the cornea and lens and converts it into the chemical energy eventually transmitted to the brain with help of the optic nerve5,6.
The aim of this paper is to provide a protocol for the dissection of corneal and retinal tissues from human ocular globes. Avoiding cross-contamination with adjacent tissues and preserving RNA integrity is of fundamental importance as such tissues are indispensable for research purposes aimed at (i) characterizing the transcriptome of the ocular tissues, (ii) isolating stem cells for regenerative medicine projects, and (iii) evaluating histological differences between tissues from normal/affected subjects. In this paper we describe the technique we currently use to remove the cornea, the choroid and retinal tissues from an ocular globe. Here we provide a detailed protocol for the dissection of the human ocular globe and the excision of corneal and retinal tissues. The accompanying video will help researchers to learn an appropriate technique for the retrieval of precious human tissues which are difficult to find regularly.

A Simplified Technique for In situ Excision of Cornea and Evisceration of Retinal Tissue from Human Ocular Globe

The paper describes a simplified technique to excise corneal and to eviscerate retinal tissues from the ocular globe of human cadaveric donors. The technique described here will help to excise good quality tissues to be used for transplantation, surgical or research purposes without damaging other tissues of the ocular globe.

Una técnica simplificada para<em&gt; In situ</em&gt; La escisión de la Córnea y la evisceración del tejido de la retina del globo ocular humano

Enucleation is the process of retrieving the ocular globe from a cadaveric donor leaving the rest of the globe undisturbed. Excision refers to the ...

Investigación

Medicina

Isolation and Excision of Murine Aorta; A Versatile Technique in the Study of Cardiovascular Disease

Cardiovascular disease is a broad term describing disease of the heart and/or blood vessels. The main blood vessel supplying the body with oxygenated blood is the aorta. The aorta may become affected in diseases such as atherosclerosis and aneurysm. Researchers investigating these diseases would benefit from direct observation of the aorta to characterize disease progression as well as to evaluate efficacy of potential therapeutics. The goal of this protocol is to describe proper isolation and excision of the aorta to aid investigators researching cardiovascular disease. Isolation and excision of the aorta allows investigators to look at gross morphometric changes as wells as allowing them to preserve and stain the tissue to look at histologic changes if desired. The aorta may be used for molecular studies to evaluate protein and gene expression to discover targets of interest and mechanisms of action. This technique is superior to imaging modalities as they have inherent limitations in technology and cost. Additionally, primary isolated cells from a freshly isolated and excised aorta can allowing researchers to perform further in situ and in vitro assays. The isolation and excision of the aorta has the limitation of having to sacrifice the animal however, in this case the benefits outweigh the harm as it is the most versatile technique in the study of aortic disease.

Pathology of the aorta can lead to severe morbidity and mortality, therefore research of disease progression and potential therapies is warranted. Here, we present a protocol to isolate and excise the murine aorta to aid researchers in their investigation of cardiovascular disease.

Aislamiento y Escisión de murino Aorta; Una técnica versátil en el Estudio de la Enfermedad Cardiovascular

Cardiovascular disease is a broad term describing disease of the heart and/or blood vessels. The main blood vessel supplying the body with oxygenated ...

Human Brown Adipose Tissue Depots Automatically Segmented by Positron Emission Tomography/Computed Tomography and Registered Magnetic Resonance Images

Reliably differentiating brown adipose tissue (BAT) from other tissues using a non-invasive imaging method is an important step toward studying BAT in humans. Detecting BAT is typically confirmed by the uptake of the injected radioactive tracer 18F-Fluorodeoxyglucose (18F-FDG) into adipose tissue depots, as measured by positron emission tomography/computed tomography (PET-CT) scans after exposing the subject to cold stimulus. Fat-water separated magnetic resonance imaging (MRI) has the ability to distinguish BAT without the use of a radioactive tracer. To date, MRI of BAT in adult humans has not been co-registered with cold-activated PET-CT. Therefore, this protocol uses 18F-FDG PET-CT scans to automatically generate a BAT mask, which is then applied to co-registered MRI scans of the same subject. This approach enables measurement of quantitative MRI properties of BAT without manual segmentation. BAT masks are created from two PET-CT scans: after exposure for 2 hr to either thermoneutral (TN) (24 &#176;C) or cold-activated (CA) (17 &#176;C) conditions. The TN and CA PET-CT scans are registered, and the PET standardized uptake and CT Hounsfield values are used to create a mask containing only BAT. CA and TN MRI scans are also acquired on the same subject and registered to the PET-CT scans in order to establish quantitative MRI properties within the automatically defined BAT mask. An advantage of this approach is that the segmentation is completely automated and is based on widely accepted methods for identification of activated BAT (PET-CT). The quantitative MRI properties of BAT established using this protocol can serve as the basis for an MRI-only BAT examination that avoids the radiation associated with PET-CT.

The method presented here uses 18F-Fluorodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET-CT) and fat-water separated magnetic resonance imaging (MRI), each scanned following 2 hr exposure to thermoneutral (24 &#176;C) and cold conditions (17 &#176;C) in order to map brown adipose tissue (BAT) in adult human subjects.

Depósitos de tejido adiposo humano Marrón segmentada automáticamente por Tomografía por Emisión de Positrones / Tomografía Computada y Resonancia Registrado Imágenes Magnéticas

Reliably differentiating brown adipose tissue (BAT) from other tissues using a non-invasive imaging method is an important step toward studying BAT in ...

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 ...

Assessment of Human Adipose Tissue Microvascular Function Using Videomicroscopy

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. It is hypothesized that pro-atherogenic mediators released from fat tissues particularly in association with central/visceral adiposity may promote pathogenic vascular changes locally and systemically, and the notion that cardiovascular disease may be the consequence of adipose tissue dysfunction continues to evolve. Here, we describe a unique method of videomicroscopy that involves analysis of vasodilator and vasoconstrictor responses of intact small human arterioles removed from the adipose depot of living human subjects. Videomicroscopy is used to examine functional properties of isolated microvessels in response to pharmacological or physiological stimuli using a pressured system that mimics in vivo conditions. The technique is a useful approach to gain understanding of the pathophysiology and molecular mechanisms that contribute to vascular dysfunction locally within the adipose tissue milieu. Moreover, abnormalities in the adipose tissue microvasculature have also been linked with systemic diseases. We applied this technique to examine depot-specific vascular responses in obese subjects. We assessed endothelium-dependent vasodilation to both increased flow and acetylcholine in adipose arterioles (50 - 350 &#181;m internal diameter, 2 - 3 mm in length) isolated from two different adipose depots during bariatric surgery from the same individual. We demonstrated that arterioles from visceral fat exhibit impaired endothelium-dependent vasodilation compared to vessels isolated from the subcutaneous depot. The findings suggest that the visceral microenvironment is associated with vascular endothelial dysfunction which may be relevant to clinical observation linking increased visceral adiposity to systemic disease mechanisms. The videomicroscopy technique can be used to examine vascular phenotypes from different fat depots as well as compare findings across individuals with different degrees of obesity and metabolic dysfunction. The method can also be used to examine vascular responses longitudinally in response to clinical interventions.

Videomicroscopy systems are used to examine functional properties of isolated adipose tissue arterioles in response to physiological and pharmacological stimuli. This technique can be used to examine microvascular phenotypes in different adipose tissue domains in obese humans.

Evaluación de la función Microvascular del tejido adiposo humano mediante videomicroscopía

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. ...

Vessel-sparing Excision and Primary Anastomosis

Urethroplasty is considered to be the standard treatment for urethral strictures since it provides excellent long-term success rates. For isolated short bulbar or posterior urethral strictures, urethroplasty by excision and primary anastomosis (EPA) is recommended. As EPA only requires the excision of the narrowed segment and the surrounding spongiofibrosis, a full-thickness transection of the corpus spongiosum, as performed in the traditional transecting EPA (tEPA), is usually unnecessary. Jordan et al. introduced the idea of a vessel-sparing approach in 2007, aiming to reduce surgical trauma, especially to the dual arterial blood supply of the urethra, and, thus, potentially reducing the risk of postoperative erectile dysfunction or glans ischemia. This approach could also be beneficial for subsequent urethral interventions such as redo urethroplasty using a free graft, in which a well-vascularized graft bed is imperative. Nevertheless, these potential benefits are only assumptions as prospective studies comparing the functional outcome of both techniques with validated questionnaires are currently lacking. Moreover, vessel-sparing EPA (vsEPA) should at least be able to provide similar surgical outcomes as tEPA. The aim of this paper is to give an elaborate, step-by-step overview of how to manage patients with isolated short bulbar or posterior urethral strictures with vsEPA. The main objective of this manuscript is to outline the surgical technique and to report the representative surgical outcome. A total of 117 patients were managed according to the described protocol. The analysis was performed on the entire patient cohort and on the bulbar (n = 91) and posterior (n = 26) vsEPA group separately. Success rates were 93.4% and 88.5% for the bulbar and posterior vsEPA, respectively. To conclude, vsEPA, as outlined in the protocol, provides excellent success rates with low complication rates for isolated short bulbar and posterior urethral strictures.

Here, we present an elaborate and efficient protocol to treat isolated short bulbar or posterior urethral strictures with vessel-sparing excision and primary anastomosis.

La supresión conservadora de vasos y Anastomosis primaria

Urethroplasty is considered to be the standard treatment for urethral strictures since it provides excellent long-term success rates. For isolated short ...

Electromagnetic Navigation Transthoracic Nodule Localization for Minimally Invasive Thoracic Surgery

The increased use of chest computed tomography (CT) has led to an increased detection of pulmonary nodules requiring diagnostic evaluation and/or excision. Many of these nodules are identified and excised via minimally invasive thoracic surgery; however, subcentimeter and subsolid nodules are frequently difficult to identify intra-operatively. This can be mitigated by the use of electromagnetic transthoracic needle localization. This protocol delineates the step-by-step process of electromagnetic localization from the pre-operative period to the postoperative period and is an adaptation of the electromagnetically guided percutaneous biopsy previously described by Arias et al. Pre-operative steps include obtaining a same day CT followed by the generation of a three-dimensional virtual map of the lung. From this map, the target lesion(s) and an entry site are chosen. In the operating room, the virtual reconstruction of the lung is then calibrated with the patient and the electromagnetic navigation platform. The patient is then sedated, intubated, and placed in the lateral decubitus position. Using a sterile technique and visualization from multiple views, the needle is inserted into the chest wall at the prechosen skin entry site and driven down to the target lesion. Dye is then injected into the lesion and, then, continuously during needle withdrawal, creating a tract for visualization intra-operatively. This method has many potential benefits when compared to the CT-guided localization, including a decreased radiation exposure and decreased time between the dye injection and the surgery. Dye diffusion from the pathway occurs over time, thereby limiting intra-operative nodule identification. By decreasing the time to surgery, there is a decrease in wait time for the patient, and less time for dye diffusion to occur, resulting in an improvement in nodule localization. When compared to electromagnetic bronchoscopy, airway architecture is no longer a limitation as the target nodule is accessed via a transparenchymal approach. Details of this procedure are described in a step-by-step fashion.

Presented here is a protocol for lung nodule localization using dye marking via electromagnetically navigated transthoracic needle access. The technique described here can be accomplished in the peri-operative period to optimize nodule localization and to successful resection when performing minimally invasive thoracic surgery.

Localización de nódulos transtorácicos de navegación electromagnética para cirugía torácica mínimamente invasiva

The increased use of chest computed tomography (CT) has led to an increased detection of pulmonary nodules requiring diagnostic evaluation and/or ...

Murine Appendectomy Model of Chronic Colitis Associated Colorectal Cancer by Precise Localization of Caecal Patch

The human appendix has been recently implicated to play important biological roles in the pathogenesis of various complex diseases, such as colorectal cancer, inflammatory bowel disease, and Parkinson&#8217;s disease. To study the function of the appendix, a gut disease-associated murine appendectomy model has been established and its step-by-step protocol is described here. This report introduces a facile protocol for caecal patch removal in mice followed by the chemical induction of chronic colitis-associated colorectal cancer using a combination of dextran sulfate sodium (DSS) and azoxymethane (AOM). IgA specific cells and IgA concentration were significantly reduced upon removal of the caecal patch in male C57BL/6 mice&#160;compared to those in the sham group. Simultaneously administering 2% DSS and AOM resulted in nearly 80% mice survival in both sham and appendectomy groups without significant body weight loss. Histological results confirmed colonic inflammation and different degrees of adenocarcinoma. This model can be used for the study of the functional role of the appendix in maintaining gut microbiota homeostasis and pathogenesis of gut colitis and malignancies, as well as for the potential development of drug targeting therapies.

The presented protocol describes a facile surgical removal of the appendix (caecal patch) in a mouse followed by the induction of inflammatory bowel disease-associated colorectal cancer. This murine appendectomy model enables investigation of the biological role of the appendix in the pathogenesis of human gastrointestinal disease.

Modelo de apendicectomía murine del cáncer colorrectal asociado a la colitis crónica por localización precisa del parche de Caecal

The human appendix has been recently implicated to play important biological roles in the pathogenesis of various complex diseases, such as colorectal ...

Inguinal Subcutaneous White Adipose Tissue (ISWAT) Transplantation Model of Murine Islets

Pancreatic islet transplantation is a well-established therapeutic treatment for type 1 diabetes. The kidney capsule is the most commonly used site for islet transplantation in rodent models. However, the tight kidney capsule limits the transplantation of sufficient islets in large animals and humans. The inguinal subcutaneous white adipose tissue (ISWAT), a new subcutaneous space, was found to be a potentially valuable site for islet transplantation. This site has better blood supply than other subcutaneous spaces. Moreover, the ISWAT accommodates a larger islet mass than the kidney capsule, and transplantation into it is simple. This manuscript describes the procedure of mouse islet isolation and transplantation in the ISWAT site of syngeneic diabetic mouse recipients. Using this protocol, murine pancreatic islets were isolated by standard collagenase digestion and a basement membrane matrix hydrogel was used for fixing the purified islets in the ISWAT site. The blood glucose levels of the recipient mice were monitored for more than 100 days. Islet grafts were retrieved at day 100 after transplantation for histological analysis. The protocol for islet transplantation in the ISWAT site described in this manuscript is simple and effective.

Inguinal Subcutaneous White Adipose Tissue ISWAT Transplantation Model of Murine Islets

In this protocol, a method of murine islet isolation and transplantation into the inguinal subcutaneous white adipose tissue is described. Isolated syngeneic murine islets are transplanted into a murine recipient using a basement membrane hydrogel. The blood glucose level of the recipients is monitored, and histology analysis of the islet grafts is performed.

Modelo de trasplante de tejido adiposo blanco subcutáneo inguinal (ISWAT) de islotes murinos

Pancreatic islet transplantation is a well-established therapeutic treatment for type 1 diabetes. The kidney capsule is the most commonly used site for ...

Abordaje posteromedial doble artroscópico para la extirpación de quistes de PCL

Arthroscopic Excision of Posterior Cruciate Ligament Cysts Using a Double Posteromedial Approach

Cruciate ligament cysts of the knee are a rare condition. Posterior cruciate ligament cysts of the knee are less common than anterior cruciate ligament cysts. In patients with asymptomatic isolated cruciate ligament cysts of the knee, conservative treatment is recommended. Symptomatic cruciate ligament cysts of the knee are mostly manifested as knee hyperflexion pain, straightening pain, knee discomfort after standing for a long time or walking for a long time, etc., which seriously affects the quality of life, surgical treatment can be performed. The surgical treatments can be divided into ultrasound-guided cyst puncture and fluid extraction procedure and arthroscopic cystectomy. Cysts are mostly lobulated with a multi-layer cyst wall, cyst fluid extraction does not remove the cyst wall completely but simply extracts cyst fluid, leading to a high recurrence rate. Arthroscopic surgery can completely remove the cyst wall with little trauma, a low recurrence rate, and fast postoperative recovery, so arthroscopic resection is the most common and preferred method of treatment. Since posterior cruciate ligament cysts mostly occur posterior to the ligament, we remove the cyst wall by adding a double posteromedial approach to the knee joint, and the cyst wall is removed under direct vision, which is simple to operate, the cyst wall is completely cleared, the trauma is small, the postoperative recovery is fast, and there is no recurrence. Here, 8 posterior cruciate ligament cysts were removed with complete postoperative symptom relief, no surgical complications, and no recurrence at 1-year follow-up.

Autor destacado: Doble abordaje posteromedial para el tratamiento de quistes del ligamento cruzado posterior

We present a surgical approach to treat posterior cruciate ligament cysts by an arthroscopic double posteromedial approach.

Escisión artroscópica de quistes del ligamento cruzado posterior mediante doble abordaje posteromedial

Cruciate ligament cysts of the knee are a rare condition. Posterior cruciate ligament cysts of the knee are less common than anterior cruciate ligament ...