Algunas de las figuras de este manuscrito fueron generosamente proporcionadas por el laboratorio de Bart O. Williams. Los autores son apoyados por el Instituto Nacional del Cáncer otorga U54CA143803, CA163124, CA093900, CA143055 Y.

Este protocolo se reúne y sigue las directrices establecidas por el Comité Institucional de Cuidado y Uso de Animales de la Universidad Johns Hopkins. 1. micro-disección de una próstata de ratón <ol><li> La eutanasia a los ratones por asfixia con dióxido de carbono o un método alternativo autorizado conforme a las directrices para el cuidado y uso de animales institucionales. </li><li> Pin el ratón en una posición supina a una tabla de disección por poner un pasador a través de cada una de las cuatro patas. </li><li> Pulverizar el abdomen con 70% de etanol. </li><li> Mantenga la piel de la parte inferior del abdomen con unas pinzas, y usar las tijeras para cortar a través de la piel y el peritoneo aproximadamente 1 cm por delante del orificio del pene, cuidado de no cortar cualquier órgano. </li><li> Cortar la piel y el peritoneo de la parte inferior del abdomen, hasta ambos lados del abdomen a la caja torácica, la exposición de la cavidad peritoneal. </li><li> Identificar la vejiga y el tracto urogenital, y exponerlos moviendo suavemente los órganos de grasa y otros a un lado. </li><li> Usiunas pinzas de agarre Ng, los conductos deferentes en la base cerca de la uretra, y arrancarla. </li><li> Repita con el conducto deferente opuestos. </li><li> Con unas pinzas, agarre cuidadosamente la vejiga y tire hacia arriba, mientras que al mismo tiempo el uso de tijeras para cortar a través de la uretra por debajo de la vejiga y la próstata ventral (aproximadamente 1 cm por debajo de la base de la vejiga). Nota: A medida que la vejiga se detuvo, el (la vejiga que contiene, una sección de la uretra, todos los lóbulos de la próstata y las vesículas seminales) todo el tracto urogenital vendrá con él. Puede haber un poco de resistencia. </li><li> Coloque el tracto urogenital (UGT) en una placa de Petri que contiene 60 a 100 mm aproximadamente 5-10 ml de fosfato salino tamponado (PBS). </li><li> Realizar la parte restante del protocolo bajo un microscopio de disección. Use dos pinzas finas, una en cada mano. Mantenga la pinza &quot;como un lápiz&quot; y descansar los antebrazos en la mesa de trabajo con el fin de mantener el equilibrio ellos. </li><li> Utilice unas pinzas finas para posicionar el UGT de tal manera que la vejiga estáen la parte superior, la uretra está apuntando hacia abajo, y las vesículas seminales están colocados a ambos lados de la vejiga. </li><li> Agarre la uretra con uno fórceps y con cuidado tire de la grasa con los otros fórceps sin que se rompa la basura cualquier tejido de la próstata. Nota: Fat aparecerá relativa &quot;brillante&quot; para el tejido circundante. </li><li> Una vez que se elimina la grasa, el uso de fórceps para rasgar el tejido conectivo entre los dos lóbulos ventrales de la próstata y separarlos. </li><li> Para eliminar uno de los lóbulos ventrales, utilice uno de fórceps para agarrar un solo lóbulo ventral cerca de la punta, y el uso de las otras pinzas para agarrar ese lóbulo en la base lo más cercano a la uretra como sea posible. Mientras que aún agarrando la base del lóbulo, utilizar las otras pinzas para agarrar la uretra. Tire del lóbulo lejos de la uretra con un movimiento firme y lisa. Asegúrese de que se mantiene sin tejido de la próstata unido a la uretra. Coloque el lóbulo en el medio apropiado, dependiendo de la siguiente etapa experimental. <ol><li> Para fijar y parafina incrustar los samplio para el análisis histológico, colocar el lóbulo en 10% de formalina tamponada neutra (NBF) 8. </li><li> Coloque el lóbulo en medio de congelación, tales como PTU, para la crioconservación y el corte 8. </li><li> Coloque el lóbulo en PBS frío para el aislamiento de células individuales para la cultura o citometría de flujo 9. </li></ol></li><li> Repita el paso 1.15 con el otro lóbulo ventral. </li><li> Para eliminar uno de los lóbulos anteriores, el uso de fórceps para tirar suavemente del lóbulo lejos de la vesícula seminal, teniendo cuidado de no perforar la vesícula seminal. Una vez que el lóbulo es independiente de la vesícula seminal, y eliminar de la misma manera como el lóbulo ventral en el paso 1,15. </li><li> Repita el paso 1.17 con el otro lóbulo anterior. </li><li> Da la vuelta al tracto urogenital restante sobre tal que la próstata dorsal es visible. </li><li> Para eliminar uno de los lóbulos dorsolaterales, el uso de fórceps para rasgar el tejido conectivo entre los dos lóbulos dorsolaterales y también el tejido conectivo entre los lóbulos y tél región posterior de las vesículas seminales. Retire el lóbulo de una manera similar como el lóbulo ventral en el paso 1,15. </li><li> Repita el paso 1.20 con el otro lóbulo dorsolateral. </li></ol> 2. La castración quirúrgica Nota: Esta es una cirugía de supervivencia, por lo que la asepsia, anestesia y el tratamiento del dolor son importantes para la realización ética y exitosa de este protocolo. Siga todas las directrices para el cuidado y uso de animales institucionales. Utilice una fuente de calor externa, tal como una manta de agua de recirculación, durante el procedimiento quirúrgico para prevenir la hipotermia. No deje un animal sin vigilancia hasta que se haya recuperado el conocimiento suficiente para mantener decúbito esternal. No devuelva un animal que ha sido sometido a una cirugía para la compañía de otros animales hasta que esté completamente recuperado. Autoclave todos los instrumentos quirúrgicos antes de su uso en cirugía. <ol><li> En una superficie limpia y estéril anestesiar al ratón usando las medidas apropiadas (por ejemplo, 2% de isoflurano inhalado) y la posición de la mOuse en una posición supina. Nota: los métodos de anestesia alternativos, como la ketamina, también se pueden utilizar, dependiendo del protocolo de animales aprobado. </li><li> Comprobar los reflejos del ratón apretando los dedos de los pies. Si el ratón no reacciona, proceder al siguiente paso. Si no reacciona, espere 1-3 minutos, y puedes volver a intentarlo. Afeitar el área quirúrgica con una maquinilla de afeitar eléctrica. </li><li> Asépticamente preparar el abdomen del ratón usando alternando matorrales de etanol al 70% y el yodo o los procedimientos recomendados por el personal veterinario institucional o IACUC. </li><li> Con un bisturí estéril practicar una incisión vertical de 1 cm a través de la piel en la línea media del abdomen inferior, de aproximadamente 1,5 cm por delante del pene. </li><li> Hacer una pequeña incisión (&lt;1 cm) a través del peritoneo, cuidado de no cortar cualquier órgano. </li><li> El uso de pinzas estériles, agarre el borde del corte del peritoneo y levantarla con cuidado a fin de ser capaz de ver la cavidad peritoneal por debajo. </li><li> El uso de otro pinzas estériles, meter la mano en THe cavidad peritoneal y el agarre sea la almohadilla de grasa de testículo izquierdo o derecho, lateral a la vejiga. Tire de la almohadilla de grasa a través de la abertura en el peritoneo y la piel hasta que el testículo sale así. Tenga cuidado de no lesionar otros tejidos u órganos durante este paso. </li><li> Con un marcador de cauterio, cortar a través de la almohadilla de grasa que mantiene los testículos. Cortar lentamente a través de la arteria testicular con el lápiz cauterio Nota: Si la arteria testicular se corta demasiado rápido, puede haber sangrado excesivo y el ratón pueden tener que ser sacrificados. </li><li> Una vez que se cortan la almohadilla de grasa y la arteria, retire la almohadilla de testículo y grasa. </li><li> Repita los pasos 2.6-2.9 con el otro testículo. Nota: Un método alternativo para la eliminación de los testículos es para ligar quirúrgicamente la arteria testicular, seguido por el corte del recipiente con unas tijeras. </li><li> Suturar el peritoneo se cerró con sutura absorbible. </li><li> Grapa la piel se cerró con grapas para heridas quirúrgicas. </li><li> Administrar un analgésico para controlar el dolor, y el lugarel ratón en una jaula limpia en una jaula más caliente durante 5-10 minutos. Monitorear el ratón para detectar signos de dolor o sangrado. </li></ol>

<ol>
	<li>Siegel, R. L., Miller, K. D., Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+statistics.">Cancer statistics.</a> CA Cancer J Clin. 65 (1), 5-29 (2015).</li><li>Roehrborn, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathology+of+benign+prostatic+hyperplasia.">Pathology of benign prostatic hyperplasia.</a> Int J Impot Res. 20 (suppl 3), 11-18 (2008).</li><li>Valkenburg, K. C., Williams, B. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+prostate+cancer.">Mouse models of prostate cancer.</a> Prostate Cancer. , 895238 (2011).</li><li>Shappell, S. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prostate+pathology+of+genetically+engineered+mice:+definitions+and+classification.+The+consensus+report+from+the+Bar+Harbor+meeting+of+the+Mouse+Models+of+Human+Cancer+Consortium+Prostate+Pathology+Committee.">Prostate pathology of genetically engineered mice: definitions and classification. The consensus report from the Bar Harbor meeting of the Mouse Models of Human Cancer Consortium Prostate Pathology Committee.</a> Cancer Res. 64 (6), 2270-2305 (2004).</li><li>Valkenburg, K. C., Pienta, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+discovery+in+prostate+cancer+mouse+models.">Drug discovery in prostate cancer mouse models.</a> Expert Opin Drug Discov. , 1-14 (2015).</li><li>El-Alfy, M., Pelletier, G., Hermo, L. S., Labrie, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unique+features+of+the+basal+cells+of+human+prostate+epithelium.">Unique features of the basal cells of human prostate epithelium.</a> Microsc Res Tech. 51 (5), 436-446 (2000).</li><li>Prins, G. S., Huang, L., Birch, L., Pu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+estrogens+in+normal+and+abnormal+development+of+the+prostate+gland.">The role of estrogens in normal and abnormal development of the prostate gland.</a> Ann N Y Acad Sci. 1089, 1-13 (2006).</li><li>Valkenburg, K. 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=Activation+of+Wnt/beta-catenin+signaling+in+a+subpopulation+of+murine+prostate+luminal+epithelial+cells+induces+high+grade+prostate+intraepithelial+neoplasia.">Activation of Wnt/beta-catenin signaling in a subpopulation of murine prostate luminal epithelial cells induces high grade prostate intraepithelial neoplasia.</a> Prostate. 74 (15), 1506-1520 (2014).</li><li>Lukacs, R. U., Goldstein, A. S., Lawson, D. A., Cheng, D., Witte, O. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+cultivation+and+characterization+of+adult+murine+prostate+stem+cells.">Isolation, cultivation and characterization of adult murine prostate stem cells.</a> Nat Protoc. 5 (4), 702-713 (2010).</li><li>Michiel Sedelaar, J. P., Dalrymple, S. S., Isaacs, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Of+mice+and+men--warning:+intact+versus+castrated+adult+male+mice+as+xenograft+hosts+are+equivalent+to+hypogonadal+versus+abiraterone+treated+aging+human+males,+respectively.">Of mice and men--warning: intact versus castrated adult male mice as xenograft hosts are equivalent to hypogonadal versus abiraterone treated aging human males, respectively.</a> Prostate. 73 (12), 1316-1325 (2013).</li><li>Wang, X., 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+luminal+epithelial+stem+cell+that+is+a+cell+of+origin+for+prostate+cancer.">A luminal epithelial stem cell that is a cell of origin for prostate cancer.</a> Nature. 461 (7263), 495-500 (2009).</li><li>Pascal, L. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5alpha-Reductase+inhibition+coupled+with+short+off+cycles+increases+survival+in+the+LNCaP+xenograft+prostate+tumor+model+on+intermittent+androgen+deprivation+therapy.">5alpha-Reductase inhibition coupled with short off cycles increases survival in the LNCaP xenograft prostate tumor model on intermittent androgen deprivation therapy.</a> J Urol. 193 (4), 1388-1393 (2015).</li><li>Shen, M. M., Abate-Shen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+genetics+of+prostate+cancer:+new+prospects+for+old+challenges.">Molecular genetics of prostate cancer: new prospects for old challenges.</a> Genes Dev. 24 (18), 1967-2000 (2010).</li></ol>

Los autores declaran que no tienen intereses financieros en competencia.

Próstata micro-disección permite la experimentación de lóbulos específica y el análisis de la próstata de ratón (Figura 1). En modelos de ratones genéticamente modificados, los fenotipos se pueden observar en un lóbulo que no se ve en otros. Además, para el análisis histológico, este protocolo asegura que la máxima cantidad de tejido de la próstata puro puede ser seccionadas y teñidas, sin otros tejidos del tracto urogenital presentes en la sección. Por último, para los experimentos de...

La próstata es el sitio más común de cáncer en hombres en los EE.UU.. Cerca de 220.000 hombres cada año serán diagnosticados con cáncer de próstata, y aproximadamente 27.000 hombres sucumbirán a la enfermedad 1. Los hombres tienen un riesgo de 1 en 7 tiempo de vida de un diagnóstico de cáncer de próstata en los EE.UU. 1. Hiperplasia de próstata benigna (BPH), un agrandamiento no canceroso relacionada con la edad de la próstata, es también una condición generalizada, que afecta a más del 80% de los hombres de más de 80 2. Como tal, la próstata es el foco de una investigación considerable. Los modelos de ratón se han utilizado ampliamente para estudiar las enfermedades de la próstata 3. En general, la próstata del ratón es un excelente representante de la glándula humana 4, pero hay similitudes y diferencias entre el ratón y la anatomía y la fisiología de próstata humana 5. En ambas especies, la próstata se encuentra en la base de la vejiga, que rodea a la uretra. La glándula de la próstata humana es una única loser, dividido en cuatro zonas: central, de transición, periférica, y el estroma fibromuscular anterior. En contraste, la próstata de ratón consta de tres lóbulos pareadas en diferentes posiciones alrededor de la uretra: anterior, ventral y dorsolateral. A nivel celular, la diferencia principal es que la célula basal a la proporción de células luminal en humanos es de aproximadamente 1: 1, pero es 1: 4 en ratones 6. El estroma murino también es diferente en ratones en relación con los seres humanos - los seres humanos tienen más extensa del músculo liso, mientras que la capa muscular es mucho más delgada en la próstata murino. La adecuada identificación, disección, y la manipulación de la próstata del ratón son esenciales para la investigación de la próstata del ratón. Los niveles hormonales afectan drásticamente el desarrollo y la homeostasis de la glándula de la próstata en los seres humanos y ratones. Mientras que el estrógeno parece desempeñar un papel en el desarrollo de la próstata 7, la hormona más importante en la próstata es la testosterona. La testosterona es esencial para un desarrollo glandularmantenimiento d. Después de cualquiera de la castración física o química, aproximadamente el 90% de las células luminales en el apoptose adulto de próstata y la glándula reduce. Si se vuelve a introducir la testosterona a un individuo en condiciones de castración, la próstata es capaz de regenerarse a sí mismo a plena capacidad. La testosterona también parece conducir a cáncer de próstata, por lo que la terapia de privación de andrógenos es una estrategia terapéutica utilizada comúnmente. Sin embargo, muchos cánceres de próstata se vuelven resistentes a la privación de andrógenos. Además, los hombres sometidos a terapia de privación de andrógenos para el tratamiento del cáncer de próstata sometidos a ciclos de condiciones castrados y regeneradas. En el ratón, la castración quirúrgica, junto con la regeneración hormonal de la próstata mediante la reintroducción de la testosterona, es una herramienta importante con el que para estudiar la resistencia a la castración y los efectos de la bicicleta testosterona en la próstata. En este artículo, vamos a discutir y demostrar las técnicas apropiadas por los que pueda LOCATe y micro-diseccionar una próstata de ratón, así como quirúrgicamente castrar a un ratón.

<table border="0" cellpadding="0" cellspacing="0" width="741">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Surgical tools</td>
			<td style="width:145px;">Roboz</td>
			<td style="width:113px;">Various</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formaldehyde</td>
			<td>Sigma</td>
			<td>F8775</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">OCT medium</td>
			<td>VWR</td>
			<td>25608-930</td>
			<td style="width:239px;">Must be placed on dry ice to freeze, and stored at -80C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td>Life Technologies</td>
			<td align="right">10010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoflurane</td>
			<td>Johns Hopkins</td>
			<td></td>
			<td>Also available from vendors online</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cautery Pen</td>
			<td>Medline</td>
			<td>ESCT002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silk suture</td>
			<td>AD Surgical</td>
			<td>M-S330R19</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical Wound Clips</td>
			<td>Roboz</td>
			<td>RS-9265</td>
			<td></td>
		</tr>
	</tbody>
</table>

murine prostate micro-dissection and surgical castration

Mouse models are used extensively to study prostate cancer and other diseases. The mouse is an excellent model with which to study the prostate and has been used as a surrogate for discoveries in human prostate development and disease. Prostate micro-dissection allows consistent study of lobe-specific prostate anatomy, histology, and cellular characteristics in the absence of contamination of other tissues. Testosterone affects prostate development and disease. Androgen deprivation therapy is a common treatment for prostate cancer patients, but many prostate tumors become castration-resistant. Surgical castration of mouse models allows for the study of castration resistance and other facets of hormonal biology on the prostate. This procedure can be coupled with testosterone reintroduction, or hormonal regeneration of the prostate, a powerful method to study stem cell lineages in the prostate. Together, prostate micro-dissection and surgical castration opens up a multitude of opportunities for robust and consistent research of prostate development and disease. This manuscript describes the protocols for prostate micro-dissection and surgical castration in the laboratory mouse.

Todos los seis lóbulos prostáticos fueron retirados de un ratón a través de la próstata micro-disección (Figura 1). El tracto urogenital completa (UGT) se compone de todos los lóbulos de la próstata, la vejiga, vesículas seminales y la uretra (Figura 1a). El conducto deferente se une a la uretra, pero no es necesaria para la próstata micro-disección, y por lo tanto se pueden separar antes de la retirada de la UGT cortando la uretra (Fi...

Watch this Scientific Journal Video about Murine Prostate Micro-dissection and Surgical Castration at JoVE.com

Murine Prostate Micro-dissection and Surgical Castration

Este manuscrito describe los protocolos de próstata micro-disección y la castración quirúrgica en el ratón de laboratorio. También se representan los resultados representativos producidos por estos protocolos. Finalmente, se discuten las ventajas y utilización de estos protocolos.

Murino de próstata micro-disección y la castración quirúrgica

Mouse models are used extensively to study prostate cancer and other diseases. The mouse is an excellent model with which to study the prostate and has ...

murine-prostate-micro-dissection-and-surgical-castration

Research

JoVE Journal

Medicine

45.2K vistas.  Johns Hopkins University.  Este manuscrito describe los protocolos de próstata micro-disección y la castración quirúrgica en el ratón de laboratorio. También se representan los resultados representativos producidos por estos protocolos. Finalmente, se discuten las ventajas y utilización de estos protocolos. 

Video: Murine Prostate Micro-dissection and Surgical Castration

Microvascular Decompression: Salient Surgical Principles and Technical Nuances

Trigeminal neuralgia is a disorder associated with severe episodes of lancinating pain in the distribution of the trigeminal nerve. Previous reports indicate that 80-90% of cases are related to compression of the trigeminal nerve by an adjacent vessel. The majority of patients with trigeminal neuralgia eventually require surgical management in order to achieve remission of symptoms. Surgical options for management include ablative procedures (e.g., radiosurgery, percutaneous radiofrequency lesioning, balloon compression, glycerol rhizolysis, etc.) and microvascular decompression. Ablative procedures fail to address the root cause of the disorder and are less effective at preventing recurrence of symptoms over the long term than microvascular decompression. However, microvascular decompression is inherently more invasive than ablative procedures and is associated with increased surgical risks. Previous studies have demonstrated a correlation between surgeon experience and patient outcome in microvascular decompression. In this series of 59 patients operated on by two neurosurgeons (JSN and PEK) since 2006, 93% of patients demonstrated substantial improvement in their trigeminal neuralgia following the procedure&#x2014;with follow-up ranging from 6 weeks to 2 years. Moreover, 41 of 66 patients (approximately 64%) have been entirely pain-free following the operation.
In this publication, video format is utilized to review the microsurgical pathology of this disorder. Steps of the operative procedure are reviewed and salient principles and technical nuances useful in minimizing complications and maximizing efficacy are discussed.

There are many available options for management of the patient with trigeminal neuralgia. Microvascular decompression, while the most invasive of all options, is also the most effective at achieving long term remission of symptoms. Video instruction on how to maximize efficacy and minimize complications with this procedure is described.

Microvascular Descompresión: Saliente principios quirúrgicos y los matices técnicos

Trigeminal neuralgia is a disorder associated with severe episodes of lancinating pain in the distribution of the trigeminal nerve. Previous reports ...

Investigación

Medicina

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

Evaluación de la absorción de nanopartículas en los tumores en tiempo real utilizando intravital imagen

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

Un modelo murino de cáncer de próstata humano Metástasis ortotópico

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

Surgical Fixation of Sternal Fractures: Preoperative Planning and a Safe Surgical Technique Using Locked Titanium Plates and Depth Limited Drilling

Different ways to stabilize a sternal fracture are described in literature. Respecting different mechanisms of trauma such as the direct impact to the anterior chest wall or the flexion-compression injury of the trunk, there is a need to retain each sternal fragment in the correct position while neutralizing shearing forces to the sternum. Anterior sternal plating provides the best stability and is therefore increasingly used in most cases. However, many surgeons are reluctant to perform sternal osteosynthesis due to possible complications such as difficulties in preoperative planning, severe injuries to mediastinal organs, or failure of the performed method.
This manuscript describes one possible safe way to stabilize different types of sternal fractures in a step by step guidance for anterior sternal plating using low profile locking titanium plates. Before surgical treatment, a detailed survey of the patient and a three dimensional reconstructed computed tomography is taken out to get detailed information of the fracture&#8217;s morphology. The surgical approach is usually a midline incision. Its position can be described by measuring the distance from upper sternal edge to the fracture and its length can be approximated by the summation of 60 mm for the basis incision, the thickness of presternal soft tissue and the greatest distance between the fragments in case of multiple fractures.
Performing subperiosteal dissection along the sternum while reducing the fracture, using depth limited drilling, and fixing the plates prevents injuries to mediastinal organs and vessels.
Transverse fractures and oblique fractures at the corpus sterni are plated longitudinally, whereas oblique fractures of manubrium, sternocostal separation and any longitudinally fracture needs to be stabilized by a transverse plate from rib to sternum to rib. Usually the high convenience of a patient is seen during follow up as well as a precise reconstruction of the sternal morphology.

Here we present a method to stabilize sternal fractures by using locked titanium plates in a low profile design. Performing subperiosteal dissection along the sternum while reducing the fracture, using depth limited drilling, and fixing the plates provides a safe surgical way.

Fijación quirúrgica de las fracturas esternales: Planificación preoperatoria y una técnica quirúrgica segura usando placas de titanio bloqueados y limitada profundidad de perforación

Different ways to stabilize a sternal fracture are described in literature. Respecting different mechanisms of trauma such as the direct impact to the ...

A Novel Murine Model of Arteriovenous Fistula Failure: The Surgical Procedure in Detail

The arteriovenous fistula (AVF) still suffers from a high number of failures caused by insufficient remodeling and intimal hyperplasia from which the exact pathophysiology remains unknown. In order to unravel the pathophysiology a murine model of AVF-failure was developed in which the configuration of the anastomosis resembles the preferred situation in the clinical setting. A model was described in which an AVF is created by connecting the venous end of the branch of the external jugular vein to the side of the common carotid artery using interrupted sutures. At a histological level, we observed progressive stenotic intimal lesions in the venous outflow tract that is also seen in failed human AVFs. Although this procedure can be technically challenging due to the small dimensions of the animal, we were able to achieve a surgical success rate of 97% after sufficient training. The key advantage of a murine model is the availability of transgenic animals. In view of the different proposed mechanisms that are responsible for AVF failure, disabling genes that might play a role in vascular remodeling can help us to unravel the complex pathophysiology of AVF failure.

Here we present a murine model of arteriovenous fistula (AVF) failure in which a clinically relevant anastomotic configuration is incorporated. This model can be used to study the pathophysiology and to test possible therapeutic interventions.

Un Modelo Murino novela de fístula arteriovenosa Fallo: El procedimiento quirúrgico en Detalle

The arteriovenous fistula (AVF) still suffers from a high number of failures caused by insufficient remodeling and intimal hyperplasia from which the ...

Pre-clinical Orthotopic Murine Model of Human Prostate Cancer

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information about this disease. The human orthotopic prostate cancer xenograft mouse model provides a useful alternative approach for understanding the specific interactions between genetically and molecularly altered tumor cells, their organ microenvironment, and for evaluation of efficacy of therapeutic regimens. This is a well characterized model designed to study the molecular events of primary tumor development and it recapitulates the early events in the metastatic cascade prior to embolism and entry of tumor cells into the circulation. Thus it allows elucidation of molecular mechanisms underlying the initial phase of metastatic disease. In addition, this model can annotate drug targets of clinical relevance and is a valuable tool to study prostate cancer progression. In this manuscript we describe a detailed procedure to establish a human orthotopic prostate cancer xenograft mouse model.

Prostate cancer is the second most common cause of cancer-related deaths in the United States. An orthotopic cancer model provides a useful approach to understand the biology of prostate cancer and to evaluate the efficacy of therapeutic regimens. This protocol describes detailed steps necessary to establish an orthotopic prostate cancer mouse model.

Preclínica modelo murino ortotópico de cáncer de próstata humano

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information ...

Intravital Microscopy of Tumor-associated Vasculature Using Advanced Dorsal Skinfold Window Chambers on Transgenic Fluorescent Mice

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static information and is often not sufficient for a correct interpretation. Intravital evaluation, in which a process is followed in time, provides extra and often unexpected information. With the creation of transgenic animals expressing cell-specific markers and live cell tracers, improvements to imaging equipment, and the development of several imaging chambers, intravital microscopy has become an important tool to better understand biological processes. This paper describes an experimental design for the investigation of tumor vessel development and of therapeutic effects in a spatial and temporal manner. Using this setup, the stage of vessel development, tip cell and lumen formation, blood flow, extravasation, an established vascular bed, and vascular destruction can be visualized and followed. Furthermore, therapeutic effects, intratumoral fate, and the localization of chemotherapeutic compounds can also be followed.

This protocol describes the intravital imaging of transgenic mice expressing cell-specific fluorescent markers. Intravital imaging provides a non-invasive method for high-resolution observations in living animals using implantable windows through which the microscopic visualization of processes is possible. This method is especially useful to study longitudinal processes.

La microscopia intravital de la utilización de la vasculatura asociada a Tumor avanzado Dorsal cámaras de ventana pliegues cutáneos en ratones transgénicos fluorescentes

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static ...

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an otherwise dynamic system, and successive phases are easily overlooked or misinterpreted. Live-cell imaging and time-lapse microscopy, in which living cells can be followed for hours or even days in a more or less continuous fashion, are therefore very informative. The protocol described here allows for the investigation of the fate of chemotherapeutic nanoparticles after the delivery of doxorubicin (dox) in living cells. Dox is an intercalating agent that must be released from its nanocarrier to become biologically active. In spite of its clinical registration for more than two decades, its uptake, breakdown, and drug release are still not fully understood. This article explores the hypothesis that lipid-based nanoparticles are taken up by the tumor cells and are slowly degraded. Released dox is then translocated to the nucleus. To prevent fixation artifacts, live-cell imaging and time-lapse microscopy, described in this experimental procedure, can be applied.

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

Live-cell imaging is a powerful tool to visualize dynamic processes. The examination of fixed cells provides only static pictures, which can lead to misinterpretation and confusion about the process. This work presents a method to study uptake, drug release, and intracellular localization of liposomal nanoparticles in living cells.

Usando In Vitro células vivas imágenes para explorar quimioterápicos entregado por nanopartículas basadas en lípidos

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an ...

Surgical Treatment for Benign Prostatic Hyperplasia: Holmium Laser Enucleation of the Prostate (HoLEP).

Benign prostatic hyperplasia (BPH) commonly occurs among elderly males. The condition causes symptoms in the lower urinary tract, thereby reducing the quality of life. BPH includes 2 major treatments: medication and surgery. Surgical treatment is often the most effective and final intervention. Holmium laser enucleation of the prostate (HoLEP), one of the most efficient surgical procedures for BPH, is conducted transurethrally. During surgery, the most important and most difficult part is to locate the surgical capsule of the prostate, which is consistent to the idea of anatomic surgery. Different skills may be needed in treating different parts of the prostate. HoLEP tends to be efficient and safe with good hemostatic properties, and able to treat bladder calculus, which may be the complications of BPH. The technique is particularly suitable for prostate of varying volumes and sizes. Indeed, HoLEP also presents certain disadvantages, such as a long learning curve and costly equipment. Regardless, this method may be the "new standard" and the excellent method for the surgical treatment of BPH.

Surgical Treatment for Benign Prostatic Hyperplasia: Holmium Laser Enucleation of the Prostate HoLEP.

Here we present a safe and efficient protocol, holmium laser enucleation of the prostate, to treat benign prostatic hyperplasia.

Tratamiento quirúrgico de la hiperplasia prostática benigna: Holmium láser enucleación de la próstata (HoLEP).

Benign prostatic hyperplasia (BPH) commonly occurs among elderly males. The condition causes symptoms in the lower urinary tract, thereby reducing the ...

Micro-dissection of Enamel Organ from Mandibular Incisor of Rats Exposed to Environmental Toxicants

Enamel defects resulting from environmental conditions and ways of life are public health concerns because of their high prevalence. These defects result from altered activity of cells responsible for enamel synthesis named ameloblasts, which present in enamel organ. During amelogenesis, ameloblasts follow a specific and precise sequence of events of proliferation, differentiation, and death. A rat continually growing incisors is a suitable experimental model to study ameloblast activity and differentiation stages in physiological and pathological conditions. Here, we describe a reliable and consistent method to micro-dissect enamel organ of rats exposed to environmental toxicants. The micro-dissected dental epithelia contain secretion- and maturation-stage ameloblasts that may be used for qualitative experiments, such as immunohistochemistry assays and in situ hybridization, as well as for quantitative analyses such as RT-qPCR, RNA-seq, and Western blotting.

Understanding enamel formation and possible alterations requires the study of ameloblast activity. Here, we describe a reliable and consistent method to micro-dissect enamel organs containing secretion- and maturation-stage ameloblasts that may be used for further quantitative and qualitative experimental procedures.

Micro-disección del órgano del esmalte de incisivo de la mandíbula de ratas expuestas a tóxicos ambientales

Enamel defects resulting from environmental conditions and ways of life are public health concerns because of their high prevalence. These defects result ...