Los autores desean agradecer a Verlene Henry Holmes y Lindsay para la asistencia en el desarrollo de este modelo. Este trabajo fue financiado parcialmente por la Fundación James S. McDonnell (# 220020173).

1. Las líneas celulares <ol><li> U87MG células de glioma se cultivan en grandes frascos de cultivo de tejidos con DMEM-F12 suplementado con suero fetal bovino al 5% (FBS). </li><li> Las células se recogen por lavado de botellas en dos ocasiones con fosfato caliente (PBS) y la incubación a 37 ° C durante 5 minutos con 10 ml de PBS que contiene 0,25% de tripsina y EDTA al 0,05%. Una vez que las células se levantan, se colocan en un tubo de 50 ml que contiene 10 ml de medios de cultivo y se centrifuga (300 X g durante 4 minutos). </li><li> Tras el lavado, las células se resuspenden a una concentración de 10 x 10 6 / ml en medios de cultivo, lo que permitió la inoculación de 50.000 células / l 5. </li><li> Las células se mantienen en hielo hasta que la inyección intracraneal. </li></ol> 2. Guía intracraneal tornillo atornillado Este procedimiento puede ser llevado a cabo varios días antes de la inyección de las células. Todos los procedimientos descritos aquí se han llevado a cabo bajo estrictas condiciones de esterilidad. <ol><li> Ratones (BALB / c nu / nu hembra; 5-6 semanas, aproximadamente 18 g) se numeran para su identificación, peso y anestesiados con una intraperitoneal (IP) la inyección de una mezcla de ketamina (100 mg / kg) y xilazina (5 mg / kg). </li><li> La piel es limpiar con una solución de yodo y de una pequeña incisión (2-3 mm) se realiza a lo largo de la parte derecha de la línea media y anterior a la línea interaural. Esto expone a las suturas coronal y sagital del cráneo. El bregma se coloca en la unión de estos dos puntos de sutura. </li><li> El tornillo de guía de punto de entrada se marca en un punto de 2,5 mm lateral y 1 mm anterior al bregma. Este punto está situado directamente sobre el núcleo caudado 1. </li><li> Un agujero de 1 x 1 mm de profundidad se perfora con una mano estéril cabo fresa espiral a través del cráneo a la duramadre. </li><li> Un tornillo de guía esterilizada que se extiende 1,6 mm por debajo de la superficie del cráneo en la duramadre se atornilla en el agujero con un destornillador hasta que quede nivelado con el cráneo (Figura 1). </li><li> Un estilete estéril o ficticia tornillo se coloca en el orificio central de la guía de tornillo para cerrar la abertura (fig. 1B). </li><li> La herida se cierra con adhesivo tisular Vetbond (cianoacrilato de n-butilo) y los ratones se les da una inyección intraperitoneal de reversine (animales pequeños) (0,1 ml / kg) y carprofeno (5 mg/kg/100 l) para la analgesia. Los ratones se dejan de recuperar en una estera de calentamiento (36 ° C), que puede tardar hasta 20 minutos. Los ratones son frecuentemente monitoreados y observados durante este tiempo de recuperación hasta que estén plenamente conscientes. </li></ol> 3. El injerto celular intracraneal <ol><li> Una jeringa con manguito Hamilton se prepara mediante la aplicación de un anillo de plástico a la punta de la aguja que permite sólo 2 mm de la aguja para extenderse por debajo de la salida del tornillo de guía (fig. 1B). El punto final es por lo tanto, la inoculación de 3,5 mm por debajo de la superficie del cráneo. </li><li> Cuatro días después de la cirugía tornillo guía, los ratones son anestesiados de nuevo que el anterior (2,1) y una pequeña incisión sobre el tornillo de trabajo para retirar el estilete. </li><li> El manguito jeringa Hamilton se llena con 5 l de las células y en común, teniendo precaución de no crear o elaborar cualquier burbuja de aire. </li><li> La jeringa se sujeta a la bomba de perfusión y la aguja se inserta en el tornillo de guía (fig. 1C). Las células se infunden a una velocidad de 30 l por hora. </li><li> El aparato automatizado (Figura 1 D) nos ha permitido inyectar un máximo de 10 animales a la vez, con un caudal constante. Las células también se puede inyectar de forma manual mediante el uso de la jeringa Hamilton esposadas o, alternativamente, la jeringa puede ser esposado por una punta de la pipeta 200 l que se ha reducido en 3 mm para permitir que la punta de la aguja para extender más allá de la guía de tornillo de 2 mm. La inyección se debe realizar a un ritmo constante constante. </li><li> Cuando se haya completado la infusión, la jeringa se retira cuidadosamente el estilete y se sustituye en el tornillo de guía. La herida está cerrada y pegada a los ratones se les da medicamentos recuperación como antes (2,7) y les permite recuperar en una estera de calentamiento. </li></ol> 4. Desafío terapéutico <ol><li> Cuatro días después de la inoculación de células U87MG, los ratones se pesan y se dividieron aleatoriamente en los grupos control y tratamiento. </li><li> El grupo control se le da una intraperitoneal (IP) de inyección de PBS (100 l), mientras que el grupo de tratamiento es una inyección IP de AMG102 (100 mg en 100 l de PBS). </li><li> Las inyecciones se repiten cada dos días durante 14 días, un total de 6 inyecciones (Figura 2). </li><li> Después de la última inyección, los ratones son monitoreados diariamente y se pesaron cada dos días. </li><li> Cuando los ratones comenzaron a mostrar importantes disfunciones neurológicas (balance de perturbación, parálisis), deshidratación, o más del 10% de pérdida de peso o moribundos, que fueron sacrificados humanitariamente. Estos humano de punto final de día se registraron luego de la curva de supervivencia de Kaplan-Meier. </li></ol> 5. Evaluación de la eficacia terapéutica </p&gt; <ol><li> La curva de supervivencia Kaplan-Meier se ha generado utilizando GraphPad Prism 4.03 para Windows, GraphPad Software, San Diego, California, EE.UU., <a href="http://www.graphpad.com">www.graphpad.com</a> 3. Muertes o euthanizations se han marcado como eventos de punto final y anotó el 1. Los animales que se mantuvo con vida al final del experimento se registraron como no-acontecimientos y anotó como 0. </li></ol> 6. Los resultados representativos: Control de los animales comenzaron a mostrar signos de trastornos neurológicos y pérdida de peso 23 días después de la inoculación inicial de las células. Esta se retrasó significativamente en el grupo tratado AMG102 a 35 días. De hecho el día 35, el 77% (n = 7 / 9) del grupo de control habían sido sacrificados. La curva de supervivencia Kaplan-Meier muestra claramente el aumento significativo de la supervivencia en respuesta a AMG102 tratamiento (Figura 3). Al día 70, el 88% de los ratones de control había sido sacrificado (n = 8 / 9) en comparación con el 37% (n = 3 / 8) de AMG102 grupo tratado. El análisis histológico de los cerebros confirmó que todos los 9 animales del grupo control desarrollaron tumores en comparación con sólo 3 de los 8 AMG102 ratones tratados (Figura 4). <img alt="Figura 1" src="/files/ftp_upload/3157/3157fig1.jpg" /> Figura 1: Imagen (A) muestra el tornillo guía intracraneal colocado en un área situada directamente sobre el núcleo caudado. El manguito jeringa Hamilton (pre-cargado con células) se coloca en el interior del tornillo de guía para que 2 mm de la aguja se extiende más allá de la guía y las células se inyectan 3,5 mm en el interior del núcleo caudado. La jeringa más tarde se extrae y se reemplaza por un mandril (B). El dispositivo de infusión automática (C) puede inyectar hasta 10 animales a la vez. Aquí se muestran cinco inyecciones simultáneas (D). <img alt="Figura 2" src="/files/ftp_upload/3157/3157fig2.jpg" /> Figura 2: Esquema del protocolo del estudio. Las células fueron inyectadas en el día 0 con el tratamiento a partir del día 4. Los animales control recibieron una inyección de PBS (100 l) IP, mientras que el grupo de tratamiento recibió una inyección de AMG102 (100 mg/100 l) IP. Estas inyecciones se administraron en días alternos durante 10 días. Un total de 6 inyecciones se administraron. Los animales fueron controlados después de 70 días para detectar signos de trastornos neurológicos y físicos. <img alt="Figura 3" src="/files/ftp_upload/3157/3157fig3.jpg" /> Figura 3: curvas de Kaplan-Meier comparando el control y AMG102 los ratones tratados. Significativa en la supervivencia se obtuvo tras el tratamiento con AMG102 (p = 0,005). <img alt="Figura 4" src="/files/ftp_upload/3157/3157fig4.jpg" /> Figura 4: Microfotografías de las secciones del cerebro coronal que demuestra el desarrollo de tumores en los animales control (A) y no el desarrollo de tumores cerebrales en los animales tratados AMG102 (B). Barra de escala es de 1 mm.

<ol>
	<li>Lal, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+implantable+guide-screw+system+for+brain+tumor+studies+in+small+animals.">An implantable guide-screw system for brain tumor studies in small animals.</a> J Neurosurg. 92, 326-333 (2000).</li><li>Jun, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMG+102,+a+fully+human+anti-hepatocyte+growth+factor/scatter+factor+neutralizing+antibody,+enhances+the+efficacy+of+temozolomide+or+docetaxel+in+U-87+MG+cells+and+xenografts.">AMG 102, a fully human anti-hepatocyte growth factor/scatter factor neutralizing antibody, enhances the efficacy of temozolomide or docetaxel in U-87 MG cells and xenografts.</a> Clin Cancer Res. 13, 6735-6742 (2007).</li><li>Pillay, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+plasticity+of+oncogene+addiction:+implications+for+targeted+therapies+directed+to+receptor+tyrosine+kinases.">The plasticity of oncogene addiction: implications for targeted therapies directed to receptor tyrosine kinases.</a> Neoplasia. 11, 448-458 (2009).</li><li>Buchanan, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiosensitization+of+glioma+cells+by+modulation+of+Met+signaling+with+the+hepatocyte+growth+factor+neutralizing+antibody.+AMG102.">Radiosensitization of glioma cells by modulation of Met signaling with the hepatocyte growth factor neutralizing antibody. AMG102.</a> J Cell Mol Med. 15, 1999-2006 (2011).</li><li>Wen, P. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+phase+II+study+evaluating+the+efficacy+and+safety+of+AMG+102+(rilotumumab)+in+patients+with+recurrent+glioblastoma.">A phase II study evaluating the efficacy and safety of AMG 102 (rilotumumab) in patients with recurrent glioblastoma.</a> Neuro Oncol. 13, 437-446 (2011).</li><li>Caretti, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+Tumor+Growth+and+Post-Irradiation+Recurrence+in+a+Diffuse+Intrinsic+Pontine+Glioma+Mouse+Model.">Monitoring of Tumor Growth and Post-Irradiation Recurrence in a Diffuse Intrinsic Pontine Glioma Mouse Model.</a> Brain Pathol. 4, 441-451 (2011).</li><li>Jallo, G. I., Volkov, A., Wong, C., Carson, B. S., Penno, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+brainstem+tumor+model:+functional+and+histopathological+characterization.">A novel brainstem tumor model: functional and histopathological characterization.</a> Childs Nerv. Syst. 22, 1519-1525 (2006).</li><li>Johns, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+efficacy+of+epidermal+growth+factor+receptor-specific+antibodies+against+gliomaxenografts+is+influenced+by+receptor+levels,+activation+status,+and+heterodimerization.">The efficacy of epidermal growth factor receptor-specific antibodies against gliomaxenografts is influenced by receptor levels, activation status, and heterodimerization.</a> Clin Cancer Res. 13, 1911-1925 (2007).</li><li>Scott, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+phase+I+clinical+trial+with+monoclonal+antibody+ch806+targeting+transitional+state+and+mutant+epidermal+growth+factor+receptors.">A phase I clinical trial with monoclonal antibody ch806 targeting transitional state and mutant epidermal growth factor receptors.</a> Proc. Natl. Acad. Sci. U. S. A. 104, 4071-4076 (2007).</li><li>Niyazi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+options+for+recurrent+malignant+glioma.">Therapeutic options for recurrent malignant glioma.</a> Radiother. Oncol. 98, 1-14 (2011).</li></ol>

Este trabajo fue apoyado por una beca de investigación de Amgen Inc.

El método de guía de tornillo intracraneal que aquí se presenta permite la creación rápida y reproducible de xenoinjertos intracraneal y en las manos de un técnico en animales entrenados, este procedimiento es muy fácil de realizar sin la necesidad de un dispositivo de estereotaxia. Nuestro método combina el uso de un tornillo de guía, para localizar con precisión la región del cerebro más adecuado para el desarrollo de tumores in vivo, y una bomba de infusión automatizado que permite la inoculación simultánea de hasta 10 animales a la vez. El tornillo de guía de procedimiento de aumento en menos de 5 minutos para llevar a cabo. La infusión de células automatizado a los 30 l / hora tiene 10 minutos para un volumen de 5 microlitros. Esto asegura una dosificación precisa a una velocidad constante y reduce el riesgo de expulsión de células en el área extra-craneal debido a un aumento de la presión intracraneal. También hemos realizado este procedimiento con 10 volúmenes de l y no han observado ningún crecimiento del tumor extra-craneal debido a un reflujo celular. A medida que la bomba de infusión automatizado permite a varios animales a inocular al mismo tiempo que podemos inyectar hasta 60 animales por hora en comparación con un procedimiento estereotáxico que se limita a unos 15 animales por hora. El paso más crítico en este procedimiento es la ubicación del tornillo de guía en el cráneo, ya que su posición determina la ubicación definitiva de la masa tumoral. Si el tornillo de guía es muy central, las células se inyectan en una región entre los dos hemisferios cerebrales, mientras que si la guía se coloca demasiado lejos hacia adelante, las células no se inyecta en el cerebro a todos. Por tanto, es esencial para ser lo más preciso posible en la localización de la guía de 2,5 mm lateral y 1 mm anterior al bregma 1. En este estudio se utilizó un tornillo de guía de acero, a pesar de tornillos de plástico guía también están disponibles para los estudios que requieren imágenes cerebrales, tales como resonancia magnética o PET 6. Incluso si la bomba de infusión automatizado es un costo prohibitivo para algunos grupos, las células todavía se puede inyectar de forma manual con más rapidez que los enfoques estereotáctica. En general, este método rápido, exacto y reproducible puede ser utilizado para estudiar el glioma, el meduloblastoma y los tumores del tronco cerebral 7. Este procedimiento se ha realizado en más de 500 ratones entre los dos laboratorios con las muertes debidas a reacciones inesperadas anestesia (&lt;1%). Los ratones que fueron atornilladas con el tornillo de guía y, posteriormente, infundidos con las células no mueren o sufren complicaciones neurológicas, como resultado del procedimiento. Los animales han sobrevivido más allá de 100 días sin ninguna complicación. A pesar de que prefiere usar un ratón sin pelo, un ratón inmuno-comprometidos con el pelo como el SCID sería tan apropiado siempre y cuando el pelo del cráneo se retiró antes de la cirugía para mantener el sitio limpio y evitar que el pelo interferir con el tornillo guía. Existen varias terapias nuevas están desarrollando actualmente para el tratamiento del glioma 10.08. Recientemente hemos demostrado que por vía subcutánea U87MG xenoinjertos glioma puede ser inhibida por el tratamiento con AMG102 3, un anticuerpo humanizado dirigido a HGF actualmente en fase de evaluación clínica en el glioma 5. Se utilizó el método descrito aquí para establecer los tumores intracraneales U87MG en los cerebros de ratones desnudos. Nuestro estudio demuestra claramente la utilidad del método y demuestra que AMG102 inhibe significativamente el crecimiento de xenoinjertos U87MG ortotópico. Vamos a seguir utilizando este modelo para determinar qué otros productos terapéuticos se pueden utilizar en combinación con AMG102 para obtener una inhibición más eficaz del crecimiento del tumor.

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th><th> Comentarios (opcional) </th></tr><tr><td> DMEM/F12 </td><td> Invitrogen </td><td> 12634010 </td><td></td></tr><tr><td> FCS </td><td> Bovogen </td><td> SFB </td><td></td></tr><tr><td> Tripsina </td><td> Invitrogen </td><td> 15050065 </td><td></td></tr><tr><td> EDTA </td><td> Sigma </td><td> E6758 </td><td></td></tr><tr><td> Balb / c nu / nu ratones </td><td> ARC Perth </td><td></td><td></td></tr><tr><td> Tornillo del soporte </td><td> Uno de los plásticos </td><td> SD-1 </td><td></td></tr><tr><td> Titular de perforación </td><td> Uno de los plásticos </td><td> DH-1 </td><td></td></tr><tr><td> Broca </td><td> Uno de los plásticos </td><td> D # 60 </td><td></td></tr><tr><td> El tornillo guía (metal) </td><td> Uno de los plásticos </td><td> C212SG </td><td></td></tr><tr><td> Tornillo de Dummy (stylus) </td><td> Uno de los plásticos </td><td> C212SD </td><td></td></tr><tr><td> Hamilton jeringa (10 l aguja fija 26g) </td><td> La gracia de la División de Ciencias Discovery </td><td> 80075 </td><td></td></tr><tr><td> PHD 22/2000 bomba de infusión </td><td> Harvard Apparatus </td><td> 70-2003 </td><td> Opcional </td></tr><tr><td> Vetbond </td><td> 3M </td><td> 1469SB </td><td></td></tr><tr><td> Thermo pad </td><td> Harvard Apparatus </td><td> 340390 </td><td></td></tr></tbody></table>

a simple guide screw method for intracranial xenograft studies in mice

El injerto de células tumorales humanas en el cerebro de ratones inmunodeprimidos es un método establecido para el estudio de los cánceres de cerebro, incluyendo glioblastoma (glioma) y el meduloblastoma. El abordaje estereotáctico utilizado sólo permite la inyección de un solo animal a la vez, es un trabajo intensivo y requiere de un equipo altamente especializado. El método de tornillo guía, inicialmente desarrollado por Lal et al., 1 fue desarrollado para eliminar los engorrosos procedimientos estereotáctica. Ahora describir un enfoque guía de tornillo modificado que es excepcionalmente rápida y segura, los cuales son fundamentales las consideraciones éticas. Cabe destacar que nuestro procedimiento ahora incorpora una bomba de infusión que permite hasta 10 animales a ser a la vez inyectados con células tumorales. Para demostrar la utilidad de este procedimiento, se estableció humanos U87MG células de glioma intracraneal como xenoinjertos en ratones, que fueron tratados con AMG102, un anticuerpo totalmente humano dirigido a HGF / factor de dispersión actualmente en fase de evaluación clínica 2-5. Inyección sistémica de AMG102 prolongó significativamente la supervivencia de todos los ratones con xenoinjertos U87MG intracraneal y dio lugar a una serie de curaciones completas. Este estudio demuestra que el método de tornillo guía es un enfoque de bajo costo, altamente reproducible para el establecimiento de xenoinjertos intracraneal. Además, proporciona un modelo relevante fisiológicos para la validación de nuevas estrategias terapéuticas para el tratamiento de los cánceres de cerebro.

Watch this Scientific Journal Video about A Simple Guide Screw Method for Intracranial Xenograft Studies in Mice at JoVE.com

A Simple Guide Screw Method for Intracranial Xenograft Studies in Mice

Con el fin de evaluar nuevos paradigmas terapéuticos para el tratamiento del glioma, fisiológicos modelos pertinentes son esenciales. Utilizamos un procedimiento de guía de tornillo implantable para el establecimiento de modelos de xenoinjerto intracraneal que es más rápido y más seguro que los enfoques estereotáctica.

Una guía sencilla Método de tornillo para intracraneal Estudios xenoinjerto en ratones

The grafting of human tumor cells into the brain of immunosuppressed mice is an established method for the study of brain cancers including glioblastoma ...

a-simple-guide-screw-method-for-intracranial-xenograft-studies-in-mice

Research

JoVE Journal

Medicine

22.9K vistas.  Monash Institute of Medical Research. Con el fin de evaluar nuevos paradigmas terapéuticos para el tratamiento del glioma, fisiológicos modelos pertinentes son esenciales. Utilizamos un procedimiento de guía de tornillo implantable para el establecimiento de modelos de xenoinjerto intracraneal que es más rápido y más seguro que los enfoques estereotáctica.

Video: A Simple Guide Screw Method for Intracranial Xenograft Studies in Mice

Epidural Intracranial Pressure Measurement in Rats Using a Fiber-optic Pressure Transducer

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and cardiac arrest. This elevation may result in further neurological injury, in the form of transtentorial herniation1,2,3,4, midbrain compression, neurological deficit or increased cerebral infarct2,4. Current therapies are often inadequate to control elevated ICP in the clinical setting5,6,7 . Thus there is a need for accurate methods of ICP measurement in animal models to further our understanding of the basic mechanisms and to develop new treatments for elevated ICP.
In both the clinical and experimental setting ICP cannot be estimated without direct measurement. Several methods of ICP catheter insertion currently exist. Of these the intraventricular catheter has become the clinical 'gold standard' of ICP measurement in humans8. This method involves the partial removal of skull and the instrumentation of the catheter through brain tissue. Consequently, intraventricular catheters have an infection rate of 6-11%9. For this reason, subdural and epidural cannulations have become the preferred methods in animal models of ischemic injury. 
Various ICP measurement techniques have been adapted for animal models, and of these, fluid-filled telemetry catheters10 and solid state catheters are the most frequently used11,12,13,14,15. The fluid-filled systems are prone to developing air bubbles in the line, resulting in false ICP readings. Solid state probes avoid this problem (Figure 1). An additional problem is fitting catheters under the skull or into the ventricles without causing any brain injury that might alter the experimental outcomes. Therefore, we have developed a method that places an ICP catheter contiguous with the epidural space, but avoids the need to insert it between skull and brain. 
An optic fibre pressure catheter (420LP, SAMBA Sensors, Sweden) was used to measure ICP at the epidural location because the location of the pressure sensor (at the very tip of the catheter) was found to produce a high fidelity ICP signal in this model. There are other manufacturers of similar optic fibre technologies13 that may be used with our methodology. Alternative solid state catheters, which have the pressure sensor located at the side of the catheter tip, would not be appropriate for this model as the signal would be dampened by the presence of the monitoring screw. 
Here, we present a relatively simple and accurate method to measure ICP. This method can be used across a wide range of ICP related animal models.

A novel technique to record the pressures within the skull is described. The minimally invasive method uses a fibre-optic pressure sensing system to accurately measure intracranial pressure (ICP) in anaesthetized rats without causing significant brain trauma. The technique may be used in a wide range of experimental models.

Medición de la presión intracraneal epidural en ratas, utilizando un transductor de presión de fibra óptica

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and ...

Investigación

Medicina

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

Un sencillo método de intubación ratón de pulmón

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

The In ovo CAM-assay as a Xenograft Model for Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions.
This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products.

The In ovo CAM-assay as a Xenograft Model for Sarcoma

The in ovo chorioallantoic membrane (CAM) is grafted with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior.

La<em&gt; En ovo</em&gt; CAM-ensayo como un Xenograftmodel para el sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for ...

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

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

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

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

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

A Simple Critical-sized Femoral Defect Model in Mice

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

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

A-crítico tamaño femoral Defecto sencillo modelo en ratones

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

Utilizing an Orally Dissolving Strip for Pharmacological and Toxicological Studies: A Simple and Humane Alternative to Oral Gavage for Animals

Prior to testing novel therapeutics in humans, short and long term preclinical (i.e., animal), repetitive pharmacological and toxicological testing is required. In most cases, the preferred route of administration is via oral delivery. At the present time, oral delivery is mostly accomplished using an oral gavage procedure, in part, because it can achieve consistent and precise dosing in the animal model. Although this method is well established it does have complications that can result in a high rate of animal attrition. To this end, the procedure introduced here describes an alternative to the oral gavage method in which the desired drug is incorporated into a tastant, orally dissolving strip (ODS) that can simply be presented to the test animal where it is then rapidly taken up with minimal manipulation of the test subject. Herein, we demonstrate that preclinical, oral drug delivery using the ODS method represents a safe, convenient, and humane alternative to oral gavage.

The following protocol describes a novel method for chronic oral drug administration using an orally dissolving strip (ODS) in lieu of the more commonly used oral gavage method. We demonstrate that preclinical, oral drug delivery using the ODS method represents a safe, convenient, and humane alternative to oral gavage.

Utilizando un Disolución de Gaza por vía oral para estudios farmacológicos y toxicológicos: una alternativa sencilla y humana a una sonda nasogástrica para Animales

Prior to testing novel therapeutics in humans, short and long term preclinical (i.e., animal), repetitive pharmacological and toxicological testing is ...

An Improved Method for Rapid Intubation of the Trachea in Mice

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity is a commonly used model to study both acute lung injury and fibrosis, and multiple methods have been developed for administering bleomycin (and other toxic agents) into the lungs. However, many of these approaches, such as transtracheal instillation, have inherent drawbacks, including the need for strong anesthetics and survival surgery. This paper reports a quick, reproducible method of intratracheal intubation that involves mild inhaled anesthesia, visualization of the trachea, and the use of a surrogate spirometer to confirm exposure. As a proof of concept, 8-12 week old C57BL/6 mice were administered either 2.0 U/kg of bleomycin or an equivalent volume of PBS, and both damage and fibrotic endpoints were measured post-exposure. This procedure allows researchers to treat a large cohort of mice in a relatively short period with little expense and minimal post-procedure care.

This article presents a rapid and simple method for administering bleomycin directly into the mouse trachea via intubation. Key advantages of this method are that it is highly reproducible, easy to master, and does not require specialized equipment or lengthy recovery times.

Un método mejorado para la rápida intubación de la tráquea en ratones

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity ...

A Method for Quantifying Upper Limb Performance in Daily Life Using Accelerometers

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. It has become important to measure a person's activities in daily life, and not just measure their capacity for activity in the structured environment of a clinic or laboratory. A wearable sensor that is now enabling measurement of daily movement is the accelerometer. Accelerometers are commercially-available devices resembling large wrist watches that can be worn throughout the day. Data from accelerometers can quantify how the limbs are engaged to perform activities in peoples' homes and communities. This report describes a methodology to collect accelerometry data and turn it into clinically-relevant information. First, data are collected by having the participant wear two accelerometers (one on each wrist) for 24 h or longer. The accelerometry data are then downloaded and processed to produce four different variables that describe key aspects of upper limb activity in daily life: hours of use, use ratio, magnitude ratio, and the bilateral magnitude. Density plots can be constructed that visually represent the data from the 24 h wearing period. The variables and their resultant density plots are highly consistent in neurologically-intact, community-dwelling adults. This striking consistency makes them a useful tool for determining if upper limb daily performance is different from normal. This methodology is appropriate for research studies investigating upper limb dysfunction and interventions designed to improve upper limb performance in daily life in people with stroke and other patient populations. Because of its relative simplicity, it may not be long before it is also incorporated in clinical neurorehabilitation practice.

This protocol describes a method to quantify upper limb performance in daily life using wrist-worn accelerometers.

Un método para cuantificar Rendimiento de las extremidades superiores en la Vida Diaria El uso de acelerómetros

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. ...

Observational Study Protocol for Repeated Clinical Examination and Critical Care Ultrasonography Within the Simple Intensive Care Studies

Longitudinal evaluations of critically ill patients by combinations of clinical examination, biochemical analysis and critical care ultrasonography (CCUS) may detect adverse events of interventions such as fluid overload at an early stage. The Simple Intensive Care Studies (SICS) is a research line that focuses on the prognostic and diagnostic value of combinations of clinical variables.
The SICS-I specifically focused on the use of clinical variables obtained within 24 h of acute admission for prediction of cardiac output (CO) and mortality. Its sequel, SICS-II, focuses on repeated evaluations during ICU admission. The first clinical examination by trained researchers is performed within 3 h after admission consisting of physical examination and educated guessing. The second clinical examination is performed within 24 h after admission and includes physical examination and educated guessing, biochemical analysis and CCUS assessments of heart, lungs, inferior vena cava (IVC) and kidney. This evaluation is repeated at days 3 and 5 after admission. CCUS images are validated by an independent expert, and all data is registered in an online secured database. Follow-up at 90 days includes registration of complications and survival status according to patient&#39;s medical charts and the municipal person registry. The primary focus of SICS-II is the association between venous congestion and organ dysfunction.
The purpose of publishing this protocol is to provide details on the structure and methods of this on-going prospective observational cohort study allowing answering multiple research questions. The design of the data collection of combined clinical examination and CCUS assessments in critically ill patients are explicated. The SICS-II is open for other centers to participate and is open for other research questions that can be answered with our data.

Structured protocols are necessary to provide answers on research questions in critically ill patients. The Simple Intensive Care Studies (SICS) provides an infrastructure for repeated measurements in critically ill patients including clinical examination, biochemical analysis and ultrasonography. SICS projects have specific focus but the structure is flexible to other investigations.

Protocolo de estudio observacional para examen clínico repetido y crítico cuidado ecografía dentro de los estudios de la Simple unidad de cuidados intensivos

Longitudinal evaluations of critically ill patients by combinations of clinical examination, biochemical analysis and critical care ultrasonography (CCUS) ...

A High-Throughput In Situ Method for Estimation of Hepatocyte Nuclear Ploidy in Mice

When the liver is injured, hepatocyte numbers decrease, while cell size, nuclear size and ploidy increase. The expansion of non-parenchymal cells such as cholangiocytes, myofibroblasts, progenitors and inflammatory cells also indicate chronic liver damage, tissue remodeling and disease progression. In this protocol, we describe a simple high-throughput approach for calculating changes in the cellular composition of the liver that are associated with injury, chronic disease and cancer. We show how information extracted from two-dimensional (2D) tissue sections can be used to quantify and calibrate hepatocyte nuclear ploidy within a sample and enable the user to locate specific ploidy subsets within the liver in situ. Our method requires access to fixed/frozen liver material, basic immunocytochemistry reagents and any standard high-content imaging platform. It serves as a powerful alternative to standard flow cytometry techniques, which require disruption of freshly collected tissue, loss of spatial information and potential disaggregation bias.

We present a robust, cost-effective, and flexible method for measuring changes in hepatocyte number and nuclear ploidy within fixed/cryopreserved tissue samples that does not require flow cytometry. Our approach provides a powerful sample-wide signature of liver cytology ideal for tracking the progression of liver injury and disease.

Un método in situ de alto rendimiento para la estimación de la ploideide nuclear de hepatocitos en ratones

When the liver is injured, hepatocyte numbers decrease, while cell size, nuclear size and ploidy increase. The expansion of non-parenchymal cells such as ...