La investigación actual está financiada por la Fundación para la Investigación Solorz-Zak.

Todos los procedimientos quirúrgicos fueron sometidos a una evaluación ética y aprobados por el Gobierno de la Alta Baviera (número de referencia: 55.2-1-54-2532.3-13-13 y -2532-136-11). Los animales son macho C57BL / 6 ratones con un peso corporal de aproximadamente 25 g. 1. Preparación de los animales <ol><li> Inducir la anestesia al poner el ratón en una cámara. Limpie la cámara con el 5% de isoflurano hasta que el animal pierda el conocimiento. </li><li> Inyectar anestésicos premezclados por vía intraperitoneal: fentanilo (0,05 mg / kg), midazolam (5 mg / kg) y medetomidina (0,5 mg / kg). Compruebe los reflejos antes y periódicamente durante el procedimiento. Reinyectar un tercio de la cantidad inicial por hora para mantener la anestesia. </li><li> Intube orotracheally con un tubo hecho de un G catéter venoso 11 20. Para la intubación fijar el animal sobre una plataforma inclinada (30 °), retraer la lengua con unas pinzas dobladas, visualizar las cuerdas vocales con un microscopio de operación e insertar el tubo en la tráqueadurante la inspiración. </li><li> Coloque el ratón en una posición boca abajo y comprobar la correcta colocación del tubo con un trozo de algodón o una microcapnograph. </li><li> Conecte el tubo de intubación al respirador. Ventilar el ratón con aire ambiente, complementado con 25% de oxígeno con una frecuencia de 180-220 respiraciones / min y un volumen de carrera de 200-250 l. </li><li> Conecte el tubo de la intubación a la microcapnograph. Mantener el final de la espiración pCO 2 a 30 mm de Hg mediante el ajuste de la frecuencia de ventilación. </li><li> Inserte una sonda de temperatura rectal y colocar el animal en una almohadilla de calefacción con el fin de mantener 37 ° C de temperatura corporal central. </li><li> Aplique el sensor pulsoxímetro anular en la pata trasera derecha. </li><li> Abra la piel sobre el cráneo con un par de tijeras. La incisión debe ser de aproximadamente 0,5 cm de largo y entre el oído y el ojo. </li><li> Diseccionar el músculo temporal izquierdo con un bisturí desde el hueso temporal. </li><li> Pegue el medidor de flujo Doppler láser (LDF) de la sonda deel hueso temporal izquierdo. Mantenga la sonda en una posición fija hasta que el pegamento se endurece. </li><li> Perforar un agujero de aproximadamente 1,5 mm de diámetro con un taladro dental en el hueso temporal izquierdo. Enfriar la médula con solución salina para evitar el daño por calor. </li><li> Inserte la sonda de ICP en la cavidad craneal. Empuje hacia delante como dorsalmente como sea posible para evitar daños en el tejido cerebral y hemorragia. </li><li> Si la sonda está en la posición correcta, corregir y sellarlo con cemento. Deje secar el cemento durante 5 min. </li><li> Ponga el ratón con cuidado a una posición supina. </li><li> Para la monitorización continua de la presión arterial, la cateterización de la arteria femoral izquierda. </li><li> Conectar el catéter femoral para el dispositivo de control de presión de la sangre. </li></ol> 2. SAH Inducción <ol><li> Abra la piel con unas tijeras desde el esternón hasta la barbilla (2 cm). Diseccionar el tejido conectivo sin rodeos y empuje las glándulas salivales a un lado. </li><li> Exponer la arteria carótida común izquierda (CCA) y movilizarla. Preservar ladel nervio vago, que se ejecuta en la misma vaina de tejido conjuntivo como el CCA. Mueva craneal y exponer y movilizar a la ACI y la ECA con la misma técnica. </li><li> Ligar el ECA en la medida de lo posible craneal. </li><li> Prefijo dos ligaduras más para el filamento de la vuelta de la ECA. </li><li> Ocluir la CCA y el ICA temporalmente con microclips. Coloque los microclips con un aplicador microclip. Asegúrese de que los clips se aplican correctamente tirando suavemente hacia atrás. </li><li> Corte un agujero para la inserción de filamentos en el ECA con una tijera de los vasos. </li><li> Inserte un filamento Prolene 5-0 con 12 mm de longitud en la LCE. </li><li> Cierre el sitio de inserción con una ligadura preestablecido. </li><li> Retire los microclips con un aplicador microclip del CCA y el ICA. </li><li> Haga avanzar el filamento con una pinza en la ICA hasta que el ICP se eleva. Un aumento repentino de la ICP indica la inducción de sangrado. </li><li> Retirar el filamento de inmediato y ligar la ECA mediante el cierre de los dos prligaduras earranged consecutivamente. Esto evita el sangrado fuera del sitio de inserción. </li><li> La sutura de la piel herida. </li><li> Monitorear los parámetros fisiológicos de los animales durante otros 20 minutos. </li><li> Retire la PIC y sondas LDF y suturar la piel herida. </li></ol> 3. Fin del Experimento <ol><li> Perfundir el animal transcardialmente con 20 ml de solución salina (temperatura ambiente) seguido de 20 ml de PFA al 4% en PBS (4 ° C). </li><li> Diseccionar el cerebro fuera del cráneo. Cortar el cráneo en la línea media y entre las cavidades orbitales. A continuación, pelar el hueso desde el cerebro. </li><li> Evaluar la distribución de la sangre en el espacio subaracnoideo. </li></ol> 4. Consideraciones en el caso de la cirugía de la supervivencia (no se muestra en el video) <ol><li> Inyectar Carprofeno (4 mg / kg por vía subcutánea) para la analgesia postoperatoria directamente después de la inducción de la anestesia. Durante el periodo de observación post-operatorio carprofeno (4 mg / kg por vía subcutánea) se inyecta cada 24 horas. </li&gt; <li> En lugar de medir la presión arterial invasiva hacen uso de un sistema de monitoreo de la presión arterial no invasiva. </li><li> Para la terminación de la inyección de anestesia agentes antagonizar por vía subcutánea: naloxona (1,2 mg / kg), flumazenilo (0,5 mg / kg) y atipamezol (2,5 mg / kg). </li><li> Retirar la intubación del animal. </li><li> Después de recuperar de los reflejos poner al animal en una cámara de precalentado. Mantenga al animal en aproximadamente 32 ° C durante 24 horas. </li><li> Los animales son controlados regularmente por la respiración espontánea y su estado general durante las primeras horas después de la cirugía. Si el tronco cerebral se ve afectada animales presentan problemas de respiración y deben ser sacrificados. </li><li> Los animales se comprobaron diariamente para su estado general y el peso corporal, así como para los déficit neurológicos y sensoriales 15. </li></ol>

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	<li>Cahill, J., Zhang, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subarachnoid+hemorrhage:+is+it+time+for+a+new+direction.">Subarachnoid hemorrhage: is it time for a new direction.</a> Stroke. 40, 86-87 (2009).</li><li>van Gijn, J., Kerr, R. S., Rinkel, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subarachnoid+haemorrhage.">Subarachnoid haemorrhage.</a> Lancet. 369, 306-318 (2007).</li><li>Lin, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+murine+model+of+subarachnoid+hemorrhage-induced+cerebral+vasospasm.">A murine model of subarachnoid hemorrhage-induced cerebral vasospasm.</a> J. Neurosci. Methods. 123, 89-97 (2003).</li><li>Altay, T., 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+novel+method+for+subarachnoid+hemorrhage+to+induce+vasospasm+in+mice.">A novel method for subarachnoid hemorrhage to induce vasospasm in mice.</a> J. Neurosci. Methods. 183, 136-140 (2009).</li><li>Feiler, S., Friedrich, B., Scholler, K., Thal, S. C., Plesnila, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardized+induction+of+subarachnoid+hemorrhage+in+mice+by+intracranial+pressure+monitoring.">Standardized induction of subarachnoid hemorrhage in mice by intracranial pressure monitoring.</a> J. Neurosci. Methods. 190, 164-170 (2010).</li><li>Bederson, J. B., Germano, I. M., Guarino, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+blood+flow+and+cerebral+perfusion+pressure+in+a+new+noncraniotomy+model+of+subarachnoid+hemorrhage+in+the+rat.">Cortical blood flow and cerebral perfusion pressure in a new noncraniotomy model of subarachnoid hemorrhage in the rat.</a> Stroke. 26, 1086-1091 (1995).</li><li>Veelken, J. A., Laing, R. J., Jakubowski, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Sheffield+model+of+subarachnoid+hemorrhage+in+rats.">The Sheffield model of subarachnoid hemorrhage in rats.</a> Stroke. 26, 1279-1283 (1995).</li><li>Kamii, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amelioration+of+vasospasm+after+subarachnoid+hemorrhage+in+transgenic+mice+overexpressing+CuZn-superoxide+dismutase.">Amelioration of vasospasm after subarachnoid hemorrhage in transgenic mice overexpressing CuZn-superoxide dismutase.</a> Stroke. 30, 867-871 (1999).</li><li>Parra, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+model+of+subarachnoid+hemorrhage+associated+cerebral+vasospasm:+methodological+analysis.">Mouse model of subarachnoid hemorrhage associated cerebral vasospasm: methodological analysis.</a> Neurol. Res. 24, 510-516 (2002).</li><li>Broderick, J. P., Brott, T. G., Duldner, J. E., Tomsick, T., Leach, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+and+recurrent+bleeding+are+the+major+causes+of+death+following+subarachnoid+hemorrhage.">Initial and recurrent bleeding are the major causes of death following subarachnoid hemorrhage.</a> Stroke. 25, 1342-1347 (1994).</li><li>Thal, S. C., Plesnila, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+intraoperative+monitoring+of+blood+pressure+and+arterial+pCO2+during+surgical+anesthesia+in+mice.">Non-invasive intraoperative monitoring of blood pressure and arterial pCO2 during surgical anesthesia in mice.</a> J. Neurosci. Methods. 159, 261-267 (2007).</li><li>Hockel, K., Trabold, R., Scholler, K., Torok, E., Plesnila, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+anesthesia+on+pathophysiology+and+mortality+following+subarachnoid+hemorrhage+in+rats.">Impact of anesthesia on pathophysiology and mortality following subarachnoid hemorrhage in rats.</a> Exp. Transl. Stroke Med. 4, 5 (2012).</li><li>Wang, Z., Schuler, B., Vogel, O., Arras, M., Vogel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+is+the+optimal+anesthetic+protocol+for+measurements+of+cerebral+autoregulation+in+spontaneously+breathing+mice?.">What is the optimal anesthetic protocol for measurements of cerebral autoregulation in spontaneously breathing mice?.</a> Exp. Brain Res. 207, 249-258 (2010).</li><li>Schwartz, A. Y., Masago, A., Sehba, F. A., Bederson, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+models+of+subarachnoid+hemorrhage+in+the+rat:+a+refinement+of+the+endovascular+filament+model.">Experimental models of subarachnoid hemorrhage in the rat: a refinement of the endovascular filament model.</a> J. Neurosci. Methods. 96, 161-167 (2000).</li><li>Feiler, S., Plesnila, N., Thal, S. C., Zausinger, S., Scholler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+matrix+metalloproteinase-9+to+cerebral+edema+and+functional+outcome+following+experimental+subarachnoid+hemorrhage.">Contribution of matrix metalloproteinase-9 to cerebral edema and functional outcome following experimental subarachnoid hemorrhage.</a> Cerebrovasc. Dis. 32, 289-295 (2011).</li></ol>

Los autores tienen nada que revelar.

Las opciones de tratamiento después de la HSA son escasos y en su mayoría ineficaces. Por lo tanto la fisiopatología del daño cerebral post-hemorrágica necesita ser entendido más con el fin de identificar nuevas dianas terapéuticas y desarrollar nuevos enfoques terapéuticos. Estandarizados y modelos animales bien reproducibles en animales modificados genéticamente, es decir, ratones, son cruciales para tales investigaciones. El modelo de CWP se ha convertido en un modelo ampliamente utilizado para HSA ...

SAH es el subtipo tiempos con el resultado menos beneficioso para los pacientes: el 40% de los pacientes mueren dentro de un mes después de que el sangrado 1 y sobrevivientes raramente tienen un desenlace clínico favorable. La gran mayoría del SAHS espontáneos (80%) son causados ​​por la ruptura de aneurismas intracraneales, que se encuentran principalmente a lo largo de la anterior y la arteria comunicante posterior, la arteria basilar, y la arteria cerebral media (ACM) 2. Tales aneurismas son difíciles de modelar en los animales y por lo tanto los modelos animales de la HSA se llevan a cabo ya sea por inyección de sangre en el espacio / ventrículos cerebrales subaracnoidea o por perforación endovascular de un recipiente subaracnoidea. Inyección de sangre autóloga en la cisterna magna es fácil de realizar y reproducible como el volumen de la sangre puede ser controlado directamente 3. Desgraciadamente, algunos aspectos de la fisiopatología HSA, por ejemplo, ellesión de los vasos, no puede ser modelado por este procedimiento. Otro enfoque técnico para la inducción de la HSA es la apertura de una vena intracisternal 4. Sin embargo, la CWP intraluminal en la sucursal de la CRM parece ser el procedimiento que los modelos de la fisiopatología de los seres humanos más estrechamente 5. El método fue desarrollado y descrito primero en ratas por Bederson y colegas y al mismo tiempo por Veelken y colegas 6,7. Más tarde, el modelo de perforación intraluminal se adaptó a ratones 8,9. Un filamento se inserta en la arteria carótida externa (ACE) y avanzó a la base del cráneo a través de la arteria carótida interna (ACI). En el punto de la MCA ramificación filamento perfora el recipiente e induce una hemorragia en el espacio subaracnoideo en la base del cráneo. Luego, la sangre se distribuye en el espacio subaracnoideo restante a lo largo de las fisuras y los vasos sanguíneos. El sangrado se detiene por la formación del coágulo en el sitio de la perforación, pero rebleedings, WHich suelen ser perjudiciales en pacientes 10, pueden ocurrir. Por consiguiente, el modelo de filamento endovascular se convirtió en un modelo de HSA ampliamente utilizado durante los últimos años. La desventaja del modelo de perforación filamento más frecuentemente mencionado es que el volumen de sangrado no se puede controlar directamente y por lo tanto puede ser variable. Esta variabilidad puede ser reducida significativamente por un control estricto de la fisiología animal y la PIC posthemorrágica. Los ratones tienen la gran ventaja de que un gran número de cepas modificadas genéticamente están disponibles. Sin embargo, debido a su pequeño tamaño de los procedimientos quirúrgicos tienden a ser más complejo que en especies más grandes, por ejemplo, ratas o conejos. Por lo tanto la reducción de escala de las técnicas desarrolladas para las ratas a los ratones a menudo no da lugar a los resultados deseados, por ejemplo, como los ratones tienen un peso corporal y el volumen de sangre muy limitadas técnicas no invasivas para la presión arterial y análisis de gases en sangre, así como para la saturación de la hemoglobina y la monitorización de la frecuencia cardíacadeberá aplicarse siempre que sea posible. Por consiguiente, el objetivo de la publicación actual es para describir el modelo de perforación filamento para HSA en ratones y para demostrar cómo este modelo se puede realizar de una manera estandarizada y altamente reproducible.

<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>operation microscope</td>
			<td>Leica</td>
			<td>KL2500</td>
			<td></td>
		</tr>
		<tr>
			<td>isoflurane vaporizer</td>
			<td>Harvard Instruments</td>
			<td>Continuous Flow Vaporizer</td>
			<td></td>
		</tr>
		<tr>
			<td>respirator</td>
			<td>Hugo Sachs</td>
			<td>Minivent 845</td>
			<td></td>
		</tr>
		<tr>
			<td>microcapnograph</td>
			<td>Hugo Sachs</td>
			<td>Type 340</td>
			<td></td>
		</tr>
		<tr>
			<td>temperature controller</td>
			<td>FHC</td>
			<td>DC Temperature Controller</td>
			<td></td>
		</tr>
		<tr>
			<td>dental drill</td>
			<td>Paggen</td>
			<td>Labset- N</td>
			<td></td>
		</tr>
		<tr>
			<td>ICP monitor</td>
			<td>Codman</td>
			<td>ICP monitor</td>
			<td></td>
		</tr>
		<tr>
			<td>blood pressure monitor</td>
			<td>AD Instruments</td>
			<td>Bridge Amp FE221</td>
			<td></td>
		</tr>
		<tr>
			<td>syringe pump</td>
			<td>World Precision Instruments</td>
			<td>SP101IZ</td>
			<td></td>
		</tr>
		<tr>
			<td>pulsoximeter</td>
			<td>Kent Scientific</td>
			<td>MouseSTAT</td>
			<td></td>
		</tr>
		<tr>
			<td>LDF</td>
			<td>Perimed</td>
			<td>Periflux 5000</td>
			<td></td>
		</tr>
		<tr>
			<td>analog data monitor</td>
			<td>AD Instruments</td>
			<td>Power Lab 16/35</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Material</td>
		</tr>
		<tr>
			<td>cement for ICP probe fixation</td>
			<td>Speiko</td>
			<td>Carboxylate cement</td>
			<td></td>
		</tr>
		<tr>
			<td>glue for LDF probe fixation</td>
			<td>Bob Smith Industries</td>
			<td>Cyanoacrylate glue (Maxi Cure and Insta Set)</td>
			<td></td>
		</tr>
		<tr>
			<td>venous catheter</td>
			<td>Johnson &#38; Johnson</td>
			<td>Jelco winged i.v. catheter; REF 4076</td>
			<td>modified intubation tube</td>
		</tr>
		<tr>
			<td>tubing for femoral catheter</td>
			<td>Smiths Medical</td>
			<td>Fine Bore Polythene Tubing; ID 0.28 mm OD 0.61 mm; REF 800/100/100</td>
			<td>cut to 30 cm length</td>
		</tr>
		<tr>
			<td>filament for vessel perforation</td>
			<td>Ethicon</td>
			<td>Prolene 5-0</td>
			<td>cut to 12 mm length</td>
		</tr>
		<tr>
			<td>surgical equipment</td>
			<td>Fine Scientific Instruments</td>
			<td>forceps medical #5, vessel scissors 8 cm, microclip 4 mm jaw</td>
			<td></td>
		</tr>
	</tbody>
</table>

a murine model of subarachnoid hemorrhage

En esta publicación de vídeo se presenta un modelo estandarizado de ratón de la hemorragia subaracnoidea (HSA). El sangrado es inducida por el Círculo endovascular de perforación Willis (CWP) y probado por (ICP) de control de presión intracraneal. De este modo se consigue una distribución homogénea en la sangre espacios subaracnoideos que rodean la circulación arterial y fisuras del cerebelo. Fisiología animal es mantenido por la intubación, ventilación mecánica y monitorización continua en línea de diversos parámetros fisiológicos y cardiovasculares: la temperatura corporal, la presión arterial sistémica, la frecuencia cardíaca y saturación de la hemoglobina. De este modo la presión de perfusión cerebral se puede monitorizar bien que resulta en un volumen de menos variable de sangre extravasada. Esto permite una mejor estandarización de perforación filamento endovascular en ratones y hace que todo el modelo altamente reproducible. Por lo tanto, es fácilmente disponible para estudios farmacológicos y fisiopatológicos en tipo salvaje y genéticaLY alterado ratones.

Mortalidad Una vez que la técnica de cirugía que se domina el procedimiento no provoca ninguna mortalidad intraoperatoria. También sangrado se puede lograr en casi todos los animales. La mortalidad postoperatoria es del 30-40%, con la mayoría de los animales mueren en el día 1 después de la cirugía (Figura 5). ICP valora después de la HSA El ICP antes del sangrado es de alrededor de 4 mmHg. Sangrado resultado en un ...

Watch this Scientific Journal Video about A Murine Model of Subarachnoid Hemorrhage at JoVE.com

A Murine Model of Subarachnoid Hemorrhage

Un modelo de ratón estándar de la hemorragia subaracnoidea por el Círculo de Willis intraluminal perforación se describe. Perforación de vasos y hemorragia subaracnoidea son monitoreados por el monitoreo de la presión intracraneal. Además diversos parámetros vitales se registran y se controlan para mantener las condiciones fisiológicas.

Un modelo murino de hemorragia subaracnoidea

In this video publication a standardized mouse model of subarachnoid  hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of  Willis ...

a-murine-model-of-subarachnoid-hemorrhage

Research

JoVE Journal

Medicine

19.5K vistas.  University of Munich Medical Center.  Un modelo de ratón estándar de la hemorragia subaracnoidea por el Círculo de Willis intraluminal perforación se describe. Perforación de vasos y hemorragia subaracnoidea son monitoreados por el monitoreo de la presión intracraneal. Además diversos parámetros vitales se registran y se controlan para mantener las condiciones fisiológicas. 

Video: A Murine Model of Subarachnoid Hemorrhage

A Low Mortality Rat Model to Assess Delayed Cerebral Vasospasm After Experimental Subarachnoid Hemorrhage

Objective: To characterize and establish a reproducible model that demonstrates delayed cerebral vasospasm after aneurysmal subarachnoid hemorrhage (SAH) in rats, in order to identify the initiating events, pathophysiological changes and potential targets for treatment.
Methods: Twenty-eight male Sprague-Dawley rats (250 - 300 g) were arbitrarily assigned to one of two groups - SAH or saline control. Rat subarachnoid hemorrhage in the SAH group (n=15) was induced by double injection of autologous blood, 48 hr apart, into the cisterna magna. Similarly, normal saline (n=13) was injected into the cisterna magna of the saline control group. Rats were sacrificed on day five after the second blood injection and the brains were preserved for histological analysis. The degree of vasospasm was measured using sections of the basilar artery, by measuring the internal luminal cross sectional area using NIH Image-J software. The significance was tested using Tukey/Kramer's statistical analysis.
Results: After analysis of histological sections, basilar artery luminal cross sectional area were smaller in the SAH than in the saline group, consistent with cerebral vasospasm in the former group. In the SAH group, basilar artery internal area (.056 &mu;m &plusmn; 3) were significantly smaller from vasospasm five days after the second blood injection (seven days after the initial blood injection), compared to the saline control group with internal area (.069 &plusmn; 3; p=0.004). There were no mortalities from cerebral vasospasm.
Conclusion: The rat double SAH model induces a mild, survivable, basilar artery vasospasm that can be used to study the pathophysiological mechanisms of cerebral vasospasm in a small animal model. A low and acceptable mortality rate is a significant criterion to be satisfied for an ideal SAH animal model so that the mechanisms of vasospasm can be elucidated 7, 8. Further modifications of the model can be made to adjust for increased severity of vasospasm and neurological exams.

Aneurysmal subarachnoid hemorrhage (SAH) is bleeding that occurs into the subarachnoid space when an aneurysm ruptures. While the morbidity and mortality from this event has been on a decline due to improved treatment approaches, the risk of vasospasm after subarachnoid hemorrhage continues to be the same as it was several years ago. The importance of establishing a comprehensive and reproducible animal model to identify initiating events of cerebral vasospasm has been the focus of research since the first use of rats in an experimental vasospasm model in 1979 by Barry et al. Early work in rats demonstrated that a single injection of autologous blood into the cisterna magna led to acute (within minutes) but not delayed cerebral vasospasm 3, 6, 14. Here we characterize a low mortality SAH rat model that results in reproducible delayed vasospasm.

Una baja mortalidad modelo de rata para evaluar vasoespasmo cerebral retardado tras la hemorragia subaracnoidea experimental

Objective: To characterize and establish a reproducible model that demonstrates delayed cerebral vasospasm after aneurysmal subarachnoid hemorrhage (SAH) ...

Investigación

Medicina

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

Modelo murino de Curación de Heridas

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

A New Murine Model of Endovascular Aortic Aneurysm Repair

Endovascular aneurysm exclusion is a validated technique to prevent aneurysm rupture. Long-term results highlight technique limitations and new aspects of Abdominal aortic aneurysm (AAA) pathophysiology. There is no abdominal aortic aneurysm endograft exclusion model cheap and reproducible, which would allow deep investigations of AAA before and after treatment. We hereby describe how to induce, and then to exclude with a covered coronary stentgraft an abdominal aortic aneurysm in a rat. The well known elastase induced AAA model was first reported in 19901 in a rat, then described in mice2. Elastin degradation leads to dilation of the aorta with inflammatory infiltration of the abdominal wall and intra luminal thrombus, matching with human AAA. Endovascular exclusion with small covered stentgraft is then performed, excluding any interactions between circulating blood and the aneurysm thrombus. Appropriate exclusion and stentgraft patency is confirmed before euthanasia by an angiography thought the left carotid artery. Partial control of elastase diffusion makes aneurysm shape different for each animal. It is difficult to create an aneurysm, which will allow an appropriate length of aorta below the aneurysm for an easy stentgraft introduction, and with adequate proximal and distal neck to prevent endoleaks. Lots of failure can result to stentgraft introduction which sometimes lead to aorta tear with pain and troubles to stitch it, and endothelial damage with post op aorta thrombosis. Giving aspirin to rats before stentgraft implantation decreases failure rate without major hemorrhage. Clamping time activates neutrophils, endothelium and platelets, and may interfere with biological analysis.

Histological and biochemical modifications after aneurysm endograft exclusion are still unclear. We describe a new model of endograft implantation on a murine aneurysm. Through thrombus analysis with and without persistant circulating blood stream interface, and new endovascular biomaterials evaluation, this model will help to better understand endovascular aneurysm exclusion pathobiology.

Un modelo murino Nueva endovascular de aneurismas aórticos

Endovascular aneurysm exclusion is a validated technique to prevent aneurysm rupture. Long-term results highlight technique limitations and new aspects of ...

Intrastriatal Injection of Autologous Blood or Clostridial Collagenase as Murine Models of Intracerebral Hemorrhage

Intracerebral hemorrhage (ICH) is a common form of cerebrovascular disease and is associated with significant morbidity and mortality. Lack of effective treatment and failure of large clinical trials aimed at hemostasis and clot removal demonstrate the need for further mechanism-driven investigation of ICH. This research may be performed through the framework provided by preclinical models. Two murine models in popular use include intrastriatal (basal ganglia) injection of either autologous whole blood or clostridial collagenase. Since, each model represents distinctly different pathophysiological features related to ICH, use of a particular model may be selected based on what aspect of the disease is to be studied. For example, autologous blood injection most accurately represents the brain&#39;s response to the presence of intraparenchymal blood, and may most closely replicate lobar hemorrhage. Clostridial collagenase injection most accurately represents the small vessel rupture and hematoma evolution characteristic of deep hemorrhages. Thus, each model results in different hematoma formation, neuroinflammatory response, cerebral edema development, and neurobehavioral outcomes. Robustness of a purported therapeutic intervention can be best assessed using both models. In this protocol, induction of ICH using both models, immediate post-operative demonstration of injury, and early post-operative care techniques are demonstrated. Both models result in reproducible injuries, hematoma volumes, and neurobehavioral deficits. Because of the heterogeneity of human ICH, multiple preclinical models are needed to thoroughly explore pathophysiologic mechanisms and test potential therapeutic strategies.

Preclinical models of intracerebral hemorrhage are utilized to mimic certain aspects of clinical disease. Thus, mechanisms of injury and potential therapeutic strategies may be explored. In this protocol, two models of intracerebral hemorrhage are described, intrastriatal (basal ganglia) injections of autologous blood or collagenase.

Inyección intraestriatal de sangre autóloga o clostridial colagenasa como modelos murinos de la hemorragia intracerebral

Intracerebral hemorrhage (ICH) is a common form of cerebrovascular disease and is associated with significant morbidity and mortality. Lack of effective ...

A Preclinical Murine Model of Hepatic Metastases

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, murine pancreatic tumor model of hepatic metastases via a hemispleen injection of syngeneic murine pancreatic tumor cells is described. This model mimics many of the clinical conditions in patients with metastatic disease to the liver. Mice consistently develop metastases in the liver allowing for investigation of the metastatic process, experimental therapy testing, and tumor immunology research.

A preclinical, murine model of hepatic metastases performed via a hemispleen injection technique.

A preclínicos modelo murino de metástasis hepática

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, ...

The Rabbit Blood-shunt Model for the Study of Acute and Late Sequelae of Subarachnoid Hemorrhage: Technical Aspects

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and controllable animal models that simulate both conditions are presently uncommon. Therefore, new models are needed in order to mimic human pathophysiological conditions resulting from SAH.
This report describes the technical nuances of a rabbit blood-shunt SAH model that enables control of intracerebral pressure (ICP). An extracorporeal shunt is placed between the arterial system and the subarachnoid space, which enables examiner-independent SAH in a closed cranium. Step-by-step procedural instructions and necessary equipment are described, as well as technical considerations to produce the model with minimal mortality and morbidity. Important details required for successful surgical creation of this robust, simple and consistent ICP-controlled SAH rabbit model are described.

The experimental intracranial pressure-controlled blood shunt subarachnoid hemorrhage (SAH) model in the rabbit combines the standard procedures &#8212; subclavian artery cannulation and transcutaneous cisterna magna puncture, which enables close mimicking of human pathophysiological conditions after SAH. We present step-by-step instructions and discuss key surgical points for successful experimental SAH creation.

El Conejo Modelo Blood-shunt para el Estudio de la aguda y secuelas tardías de Hemorragia subaracnoidea: Aspectos Técnicos

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and ...

A Murine Model of Irreversible and Reversible Unilateral Ureteric Obstruction

Obstruction of the kidney may affect native or transplanted kidneys and results in kidney injury and scarring. Presented here is a model of obstructive nephropathy induced by unilateral ureteric obstruction (UUO), which can either be irreversible (UUO) or reversible (R-UUO). In the irreversible UUO model, the ureter may be obstructed for variable periods of time in order to induce increasingly severe renal inflammation and interstitial fibrotic scarring. In the reversible R-UUO model the ureter is obstructed to induce hydronephrosis, tubular dilation and inflammation. After a suitable period of time the ureteric obstruction is then surgically reversed by anastomosis of the severed previously obstructed ureter to the bladder in order to allow complete decompression of the kidney and restoration of urinary flow to the bladder. The irreversible UUO model has been used to investigate various aspects of renal inflammation and scarring including the pathogenesis of disease and the testing of potential anti-inflammatory or anti-fibrotic therapies. The more challenging model of R-UUO has been used by some investigators and does offer significant research potential as it allows the study of inflammatory and immune processes and tissue remodeling in an injured and scarred kidney following the removal of the injurious stimulus. As a result, the R-UUO model offers investigators the opportunity to explore the resolution of kidney inflammation together with key aspects of tissue repair. These experimental models are of relevance to human disease as patients often present with obstruction of the renal tract that requires decompression and are commonly left with significant residual kidney impairment that has no current treatment options and may lead to eventual end stage kidney failure.

The murine model of irreversible unilateral ureteric obstruction (UUO) is presented together with the model of reversible UUO in which the ureteric obstruction is relieved by anastomosis of the severed ureter into the bladder. These models enable the study of renal inflammation and scarring as well as tissue remodeling.

Un modelo murino de irreversible y reversible obstrucción ureteral unilateral

Obstruction of the kidney may affect native or transplanted kidneys and results in kidney injury and scarring. Presented here is a model of obstructive ...

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 ...

Integrated Compensatory Responses in a Human Model of Hemorrhage

Hemorrhage is the leading cause of trauma-related deaths, partly because early diagnosis of the severity of blood loss is difficult. Assessment of hemorrhaging patients is difficult because current clinical tools provide measures of vital signs that remain stable during the early stages of bleeding due to compensatory mechanisms. Consequently, there is a need to understand and measure the total integration of mechanisms that compensate for reduced circulating blood volume and how they change during ongoing progressive hemorrhage. The body&#39;s reserve to compensate for reduced circulating blood volume is called the &#39;compensatory reserve&#39;. The compensatory reserve can be accurately evaluated with real-time measurements of changes in the features of the arterial waveform measured with the use of a high-powered computer. Lower Body Negative Pressure (LBNP) has been shown to simulate many of the physiological responses in humans associated with hemorrhage, and is used to study the compensatory response to hemorrhage. The purpose of this study is to demonstrate how compensatory reserve is assessed during progressive reductions in central blood volume with LBNP as a simulation of hemorrhage.

The purpose of this protocol is to demonstrate the techniques for measuring compensatory responses to reduced central blood volume using lower body negative pressure as a noninvasive experimental model of human hemorrhage which can be used to quantify the total integration of compensatory mechanisms to blood volume deficit in humans.

Las respuestas compensatorias integradas en un modelo humano de la hemorragia

Hemorrhage is the leading cause of trauma-related deaths, partly because early diagnosis of the severity of blood loss is difficult. Assessment of ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Medición de la contractilidad isométrica de la arteria mesentérica de ratón con cable miografía

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...