SN y MDD recibieron el apoyo de los NIH K08 NS083740 y el Departamento de Neurología de la UCLA. AJG reconoce el apoyo por el Dr. Miriam y la Fundación de Investigación Sheldon G. Adelson Médica y la Fundación Larry L. Hillblom. KLN agradece el apoyo del Centro de Accidentes Cerebrovasculares Asociación Americana del Corazón 14BFSC17760005 ASA-Bugher. ILL, EGS y STC fueron apoyados por el NIH R01 NS071481. JDH reconoce el apoyo de NIH K08 NS083740.

El uso de animales en este protocolo se ha realizado de acuerdo con los procedimientos aprobados por la Universidad de California en Los Ángeles Cuidado de Animales y el empleo. Nota: Comience por identificar la población objetivo murino. En estudios anteriores, de tipo salvaje ratones C57 / Bl6 solamente masculinas se han utilizado, sin embargo varios ratones transgénicos o knockout también se pueden utilizar. Tenga en cuenta que las coordenadas estereotácticas se basan en C57 / Bl6 anatomía. Se recomienda que cada usuario verificar inicialmente la localización de la carrera a la sustancia blanca. 1. Sustancia Blanca carrera de inducción - Aproximación oblicua medial <ol><li> Comience por la preparación de una pipeta de vidrio tirado usando tubo capilar 0,5 mm de tal manera que el diámetro distal es de entre 15 a 25 micras 11. </li><li> Preparar una alícuota de 10 l estéril de L-Nio (N (5) - (1) -iminoethyl-L-ornitina HCl) a 27,4 mg / ml (130 mM) en solución salina normal estéril al 0,9%. </li><li> Pre-llenar la pipeta de vidrio tiradocon un pequeño volumen de L-Nio (2-5 l) fijando el pipeta de vidrio a un tubo conectado a una línea de vacío. Acostar a la pipeta sobre la mesa de trabajo e introduzca el extremo tirado en la solución de L-Nio. <ol><li> Aplicar el vacío hasta que al menos 2 mm de la porción de 0,5 mm de la pipeta se llena. Apague el vacío y retirar la pipeta. Coloca a un lado hasta Paso 1.12. </li></ol></li><li> Coloque el ratón en una cámara de inducción e inducir anestesia del ratón usando estándar de 32% de isoflurano fluía a través de un vaporizador (5 L / min inhalado con 5 L / min de oxígeno y 0,5 L / min N 2) durante 1 min o hasta que profundamente anestesiados. Transferir el ratón para un aparato estereotáxico equipado con un microscopio estereotáctica. Proporcionar anestesia de mantenimiento utilizando 32% de isoflurano fluido a través de un vaporizador (2 L / min inhalado con 5 L / min de oxígeno y 0,5 L / min N 2) y un cono de nariz. Comprobar la profundidad de la anestesia con una pizca dedo del pie. </li><li> Ajuste el brazo de la inyección de 36 °. </li><li> Affixa sacó titular de la pipeta de vidrio al extremo distal de un sistema de inyección a presión de bajo volumen y adjuntarlo al brazo de la inyección de la configuración estereotáctica. </li><li> Escudo bigotes del animal anestesiado con vaselina y lugar artificial ungüento de lágrimas sobre ambos ojos. Preparar un campo quirúrgico estéril mediante la colocación de un campo estéril sobre la cabeza del animal con una abertura de 5 a 10 cm sobre la cabeza. Preparar una superficie quirúrgica aspetic por el afeitado de la piel que recubre el cráneo. Limpiar el cuero cabelludo con la alternancia de betadine y el 70% algodones con alcohol. </li><li> Hacer una incisión media de 1,5 cm del cuero cabelludo con unas tijeras finas estériles para exponer la superficie del cráneo. Se seca el cráneo con un hisopo de algodón estéril y usando un microscopio estereotáctica en 1-3x magnificación, retirar cualquier tejido del periostio que recubre el uso de una herramienta de punto de micro estéril. </li><li> Marcar el bregma como punto de referencia con un marcador de punta fina. </li><li> Perforar una craneotomía ellipitical 2 mm con una broca quirúrgica estéril fina stip0; comenzando posterior al bregma y que se extiende hacia delante justo a la izquierda de la línea media. Eliminar fragmentos de hueso y el tejido superior de blando, de manera que la corteza cerebral puede ser visualizado. </li><li> Mantenga el campo quirúrgico y la superficie cortical húmedo aplicando intermitentemente gotas de solución salina estéril. </li><li> Colocar una pipeta de vidrio tirado al brazo inyector del aparato estereotáxico. Alinear el extremo distal de la pipeta con la Bregma y poner a cero las coordenadas estereotácticas. </li><li> Avanzar en la pipeta a la primera anterior / posterior (A / P) y medial / lateral (M / L) coordenadas proporcionadas en la Tabla 1. </li><li> Avanzar en la pipeta a la superficie cortical y cero el ventral (V D /) medición dorsal /. </li><li> Pasar lentamente la pipeta en el cerebro hasta llegar a la primera D / V de coordenadas en la Tabla 1. </li><li> El uso de un sistema de inyección de baja presión de volumen ajustado a 20 psi durante 20 ms pulsos, inyectar 100 nl de L-Nio en el cerebro y esperar 5 minutos para prevenir el reflujohasta la pista de la pipeta. <ol><li> Utilizar una retícula calibrada en el ocular del microscopio estereotáctica y un aumento de 3 veces. </li><li> Por consiguiente, desplazar a un total de 0,100 mm 3 (0,5 mm de longitud en una pipeta 0,5 mm de diámetro, que corresponde a 100 nl) de la pipeta de vidrio tirado para cada conjunto de coordenadas. Mediante el uso de una retícula, medir y estandarizar ya que cada organizadas varía según la ampliación y las escalas utilizadas. </li><li> Para la medición exacta del volumen durante cada inyección, el enfoque de la pipeta en ángulo con el microscopio de un lado para que el menisco aire-líquido tiene una vista sagital. El menisco debe aparecer en el mismo plano focal, tanto de la pared interior y exterior de la pipeta. </li></ol></li><li> Retire lentamente la pipeta y repita los pasos 1.13-1.16.3 en el segundo y tercer conjunto de coordenadas que figuran en la Tabla 1. </li><li> Después de la inyección final, retire la pipeta y colocar la cera suficiente hueso para llenar el sitio de la craneotomía. UNpproximate los bordes de la herida del cuero cabelludo y se unen con adhesivo dérmico. </li><li> Inyectar 0,1 ml de 0,5% bupivacaína en los márgenes de la herida utilizando un 30 G aguja estéril para evitar para prevenir el dolor local asociado con la incisión del cuero cabelludo. </li><li> Devolver el animal a la vivienda y suministro de antibióticos postoperatorios (0,48 mg / ml de trimetoprim-sulfametoxazol, o 0,5 mg / ml levofloxacino) en el agua de bebida durante 5 días. </li></ol> 2. Sustancia Blanca carrera de inducción - Aproximación oblicua posterior <ol><li> Realice los pasos 1.1 a 1.12 como en el protocolo de enfoque en ángulo medial, excepto ajustar el brazo de la inyección de la configuración estereotáctica a 45 grados orientados anterior a la posterior. </li><li> Avanzar en la pipeta a la primera A / P y M / L de coordenadas que figuran en la Tabla 2. </li><li> pasos restantes completos 1.14-1.20 como en el protocolo de enfoque en ángulo lateral. </li></ol> 3. El etiquetado neuronal retrógrada <ol><li> Preparar una estéril 10alícuota l de L-Nio a 54,8 mg / ml en solución salina normal al 0,9%. </li><li> Preparar una alícuota estéril de 20% Fluororuby (o 20% de amina de dextrano biotinilado o 2% Fluorogold) en solución salina normal al 0,9%. </li><li> Diluir juntos 1: 1 para concentraciones finales de 27,4 mg / ml L-Nio y 10% Fluororuby. </li><li> Realizar el protocolo de ACV como anteriormente en las etapas 1.3-1.23. </li><li> Visualizar el trazador fluorescente de forma nativa en la sección de tejido mediante fijación por perfusión, cryosectioning y microscopía como se ha descrito previamente 8. </li></ol> 4. Procesamiento de tejidos para la inmunofluorescencia <ol><li> En un intervalo posterior al accidente cerebrovascular adecuada que varía de 3 horas a 14 días después del accidente cerebrovascular, la eutanasia a los ratones a través de la sobredosis de isoflurano o CICUAL local aprobó procedimiento. </li><li> Abrir la cavidad torácica con unas tijeras en ángulo e insertar una aguja 23 G de la mariposa en el ventrículo izquierdo. </li><li> Coloque una pequeña incisión en la aurícula derecha con unas tijeras finas para permitir una vía de salida para el líquido de perfusión. </li><li> Transcardially perfundir con 30-40 ml de solución salina tamponada con fosfato fría, seguido de 30 a 40 ml de paraformaldehído al 4% frío a una velocidad de 10 ml / min a TA. </li><li> Decapitar al ratón y eliminar el cerebro con unas tijeras estériles para abrir el cráneo por detrás y luego retire con cuidado el cráneo recubre con una espátula, colocar el cerebro en frío PFA al 4% durante 24 horas, y luego se transfieren a un 30% de sacarosa en PBS durante 48 horas . </li><li> Preparar cuarenta secciones micras flotantes usando un criostato y realizar el procesamiento de anticuerpo como se describe anteriormente 6-8. En este estudio, utilice los siguientes anticuerpos: (: 500 dilución 1); de conejo anti-neurofilamentos 200 conejo anti-vimentina (1: 500); de cabra anti-GFAP (1: 500); conejo anti-Iba-1 (1: 1.000). </li></ol> 5. Procesamiento de tejidos para la proteína o el análisis de ARN <ol><li> En un intervalo posterior al accidente cerebrovascular adecuada que varía de 3 horas a 14 días después del accidente cerebrovascular, la eutanasia mediante sobredosis de isoflurano o CICUAL local aprobó procedimiento. </li><li> decapitar alratón y eliminar el cerebro con unas tijeras estériles para abrir el cráneo posterior y retire suavemente el cráneo recubre con una espátula. </li><li> Insertar un 4 mm espátula estéril en la parte delantera del cerebro para cortar el bulbo olfatorio y los nervios ópticos. Levante suavemente el cerebro fuera de la bóveda craneal y el lugar en tampón de disección enfriado con hielo (solución salina equilibrada de 1x Hank, 25 mM HEPES-KOH, pH 7,4, glucosa 35 mM, bicarbonato de sodio 4 mM, y 0,01 mg / ml ciclohexamida). </li><li> El uso de un bloque de cerebro y cuchillas de afeitar estériles nuevas, preparar losas 2-3 mm que contienen la carrera y el lugar en el tampón de disección fría. </li><li> Bajo un microscopio de disección, identificar la materia blanca de la corteza motora subyacente en el hemisferio inyectado. A intervalos post-carrera más larga, la región puede ser identificado visualmente por necrosis focal y la palidez de la mielina. Nota: A intervalos post-ictus anteriores, la inyección de L-Nio mezcla con 1 l de 10% Fast Green puede permitir la identificación visual de la carrera ( <strong&gt; Figura 4A). </li><li> Bajo la guía de un microscopio de disección y el uso de un bisturí fresca, diseccionar cuidadosamente la región de la sustancia blanca que contiene el accidente cerebrovascular, identificado por cualquiera de etiquetado verde rápida o pérdida de tejido. Retirar la corteza suprayacente y el cuerpo estriado subyacente si lo deseas. </li></ol>

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Un número de modelos anteriores de accidente cerebrovascular subcortical se han descrito incluyendo inyecciones focales de endotelina-1 en la cápsula interna, materia blanca subcortical y el cuerpo estriado en la rata y el ratón 12 a 14 6,15. Los modelos más recientes de pequeños accidentes cerebrovasculares focales han utilizado la inyección microembolias colesterol en la arteria carótida y la oclusión 16 photothrombotic de una sola de las arteriolas penetrantes 17. C...

Stroke affecting the subcortical white matter is a common clinical entity, accounting for up to 25% of clinical strokes annually in the US 1. Ischemic damage to white matter also occurs silently at a significantly higher rate and contributes to the development of vascular dementia 2,3. Presently, patients with this form of cerebral ischemia have few, if any treatment choices. Despite the clinical importance of this disease, few clinically relevant animal models exist 4,5. 
The goal of this protocol is to produce a focal ischemic lesion within the murine white matter. This murine model of human disease allows the specific study of axonal injury response to stroke and how the cellular elements of white matter, namely oligodendrocytes and astrocytes along with axons, respond to and repair after stroke.
Previous reports have described a model of subcortical white matter stroke using endothelin-1 (ET-1) 6 that is similar to the one described here. Several key changes to the experimental protocol have been made thereby the potential uses of this model have expanded 7,8. This protocol provides a reliable and modifiable strategy to produce a focal stroke within mouse brain white matter. 
The major advantages of this model are the use of a chemical endothelial nitric oxide synthase (eNOS) inhibitor N(5)-(1)-iminoethyl-L-ornithine HCl (L-Nio) 9 with no known paracrine effects on cellular elements of white matter which had been a complication of models using endothelin-1 10. In addition, the stereotactic targeting of white matter in the mouse allows the use of any variety of transgenic or knockout strains, greatly expanding the available tools to determine the effect of stroke on brain white matter. Here, two variations on this technique are described and demonstrate some of the additional variations that can be utilized to enhance the understanding of axonal and white matter damage and repair after stroke.

<table border="0" cellpadding="0" cellspacing="0" width="725">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">L-N5-(1-Iminoethyl)ornithine, Dihydrochloride</td>
			<td style="width:204px;">Calbiochem</td>
			<td style="width:133px;">400600-20MG</td>
			<td style="width:81px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoflurane</td>
			<td>Phoenix Pharmaceutical, Inc.</td>
			<td>NDC 57319-559-06</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Capillary tubes</td>
			<td>World Precision Instruments</td>
			<td>50-821-807</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Picospritzer</td>
			<td>Parker Instrumentation</td>
			<td>Picospritzer II</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stereotactic setup</td>
			<td>Kent Scientific</td>
			<td>KSC51725</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pipette puller</td>
			<td>KOPF</td>
			<td>Model 720</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stereomicroscope SZ51</td>
			<td>Olympus</td>
			<td>88-124</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fine scissors</td>
			<td>Fine Scientific Tools</td>
			<td>14084-08</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forceps</td>
			<td>Harvard Apparatus</td>
			<td>PY2 72-8547</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Curved Forceps</td>
			<td>Harvard Apparatus</td>
			<td>PY2 72-8598</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blunt dissection tool</td>
			<td>Fine Scientific Tools</td>
			<td>10066-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Drill</td>
			<td>Dremel</td>
			<td>8220-1/28</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Drill bits</td>
			<td>Fine Scientific Tools</td>
			<td>19007-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vetbond</td>
			<td>3M</td>
			<td>1469SB&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Marcaine</td>
			<td>HOSPIRA</td>
			<td>NDC 0409-1610-50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trimethoprim-Sulfamethaxole</td>
			<td>STI Pharmacy</td>
			<td>NDC 54879-007-16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluororuby</td>
			<td>Fluorochrome Inc</td>
			<td>30mg</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td>Fisher</td>
			<td>O4042-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sucrose</td>
			<td>Fisher</td>
			<td>BP220-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryostat</td>
			<td>Leica CM3050 S</td>
			<td>14047033518</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass slides</td>
			<td>Fisher</td>
			<td>12-544-7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fast Green&#160;</td>
			<td>Sigma</td>
			<td>F7252-5G</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissection microscope</td>
			<td>Nikon</td>
			<td>SMZ1500</td>
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		<tr height="21">
			<td height="21" style="height:21px;">23 gauge butterfly needle</td>
			<td>Fisher</td>
			<td>14-840-35</td>
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		<tr height="21">
			<td height="21" style="height:21px;">10X Hank&#39;s Balanced Salt Solution</td>
			<td>Life Technologies</td>
			<td>14065056</td>
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		<tr height="21">
			<td height="21" style="height:21px;">1M HEPES-KOH, pH 7.4</td>
			<td>Affymetrix</td>
			<td>16924</td>
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			<td height="21" style="height:21px;">D-Glucose</td>
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			<td>G8270</td>
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			<td height="21" style="height:21px;">Sodium bicarbonate</td>
			<td>Sigma</td>
			<td>S5761</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cyclohexamide</td>
			<td>Sigma</td>
			<td>01810</td>
			<td></td>
		</tr>
	</tbody>
</table>

a versatile murine model of subcortical white matter stroke for the study of axonal degeneration and white matter neurobiology

Accidente cerebrovascular que afecta a las cuentas de la materia blanca de hasta el 25% de los accidentes cerebrovasculares presentaciones clínicas, se produce en silencio a velocidades que pueden ser 5-10 veces mayor, y contribuye de manera significativa al desarrollo de la demencia vascular. Pocos modelos de derrame cerebral focal materia blanca existen y esta falta de modelos apropiados ha dificultado la comprensión de los mecanismos neurobiológicos implicados en la respuesta lesión y reparación después de este tipo de accidente cerebrovascular. La principal limitación de otros modelos de accidente cerebrovascular subcortical es que no restrinjan de manera focal del infarto de la sustancia blanca o principalmente han sido validados en las especies no murinos. Esto limita la capacidad de aplicar la amplia variedad de herramientas de investigación murinos para estudiar la neurobiología de la carrera de la materia blanca. Aquí presentamos una metodología para la producción fiable de un derrame cerebral focal en la sustancia blanca murino mediante una inyección local de un inhibidor de eNOS irreversible. También presentamos algunas variaciones en el protocolo general entre ellos dos estereotáctica únicovariaciones, la localización neuronal retrógrada, así como el etiquetado de disección de tejido fresco y que en gran medida ampliar las posibles aplicaciones de esta técnica. Estas variaciones permiten múltiples enfoques para analizar los efectos neurobiológicos de esta forma común y poco estudiada de accidente cerebrovascular.

Usando el modelo presentado, la sustancia blanca subyacente extremidad anterior corteza sensoriomotora fiable puede ser objetivo. Este modelo accidente cerebrovascular inducida químicamente produce axonal focal y la pérdida de mielina, astrocitosis y microgliosis (Figura 1), como se ve típicamente en infartos lacunares humanos. Mediante el uso de tres inyecciones, un modelo de utilidad clínica se establece con deterioro temprano en las tareas motoras miembro delanter...

Watch this Scientific Journal Video about A Versatile Murine Model of Subcortical White Matter Stroke for the Study of Axonal Degeneration and White Matter Neurobiology at JoVE.com

A Versatile Murine Model of Subcortical White Matter Stroke for the Study of Axonal Degeneration and White Matter Neurobiology

Here we present methodology for the production of a focal stroke in murine white matter by local injection of an irreversible endothelial nitric oxide synthase (eNOS) inhibitor (L-Nio). Presented are two stereotactic variations, retrograde neuronal tracing, and fresh tissue labeling and dissection that expand the potential applications of this technique.

Un movimiento versátil modelo murino de la sustancia blanca subcortical para el Estudio de la degeneración axonal y la sustancia blanca Neurobiología

Stroke affecting white matter accounts for up to 25% of clinical stroke presentations, occurs silently at rates that may be 5-10 fold greater, and ...

a-versatile-murine-model-subcortical-white-matter-stroke-for-study

Research

JoVE Journal

Medicine

8.0K vistas.  University of California, Los Angeles. Here we present methodology for the production of a focal stroke in murine white matter by local injection of an irreversible endothelial nitric oxide synthase (eNOS) inhibitor (L-Nio). Presented are two stereotactic variations, retrograde neuronal tracing, and fresh tissue labeling and dissection that expand the potential applications of this technique.

Video: A Versatile Murine Model of Subcortical White Matter Stroke for the Study of Axonal Degeneration and White Matter Neurobiology

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize their blood glucose levels, islet transplantation has been proposed to be a potential treatment for type 1 diabetes 1,2. More recently, advances in human islet transplantation have further strengthened this view 1,3. However, two major limitations prevent islet transplantation from being a widespread clinical reality: (a) the requirement for large numbers of islets per patient, which severely reduces the number of potential recipients, and (b) the need for heavy immunosuppression, which significantly affects the pediatric population of patients due to their vulnerability to long-term immunosuppression. Strategies that can overcome these limitations have the potential to enhance the therapeutic utility of islet transplantation.
Islet transplantation under the mouse kidney capsule is a widely accepted model to investigate various strategies to improve islet transplantation. This experiment requires the isolation of high quality islets and implantation of islets to the diabetic recipients. Both procedures require surgical steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol. We also briefly discuss different transplantation models: syngeneic, allogeneic, syngeneic autoimmune, and allogeneic autoimmune.

Transplantation of isolated islets has been proposed to be a potential treatment for type 1 diabetes. Here we describe a method to isolate islets from mouse pancreata and transplant them to the subcapsular space of the kidney.

Un método para el aislamiento de los islotes murinos y Trasplante Renal subcapsular

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize ...

Investigación

Medicina

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Biomarcadores en un modelo animal para Revelar correlatos neurales, hematológicas y conductuales de estrés postraumático

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

A Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis

Despite the considerable progress made in the stent development in the last decades, cardiovascular diseases remain the main cause of death in western countries. Beside the benefits offered by the development of different drug-eluting stents, the coronary revascularization bears also the life-threatening risks of in-stent thrombosis and restenosis. Research on new therapeutic strategies is impaired by the lack of appropriate methods to study stent implantation and restenosis processes. Here, we describe a rapid and accessible procedure of stent implantation in mouse carotid artery, which offers the possibility to study in a convenient way the molecular mechanisms of vessel remodeling and the effects of different drug coatings.

A model of stent implantation in mouse carotid artery is described. Compared to other similar methods, this procedure is very rapid, simple and accessible, offering the possibility to study in a convenient way the vascular wall reaction to different drug-eluting stents and the molecular mechanisms of restenosis.

Un modelo murino de la implantación de un stent en la arteria carótida para el Estudio de la reestenosis

Despite  the considerable progress made in the stent development in the last decades,  cardiovascular diseases remain the main cause of death in western ...

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.

A standardized mouse model of subarachnoid hemorrhage by intraluminal Circle of Willis perforation is described. Vessel perforation and subarachnoid bleeding are monitored by intracranial pressure monitoring. In addition various vital parameters are recorded and controlled to maintain physiologic conditions.

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 ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Excitotóxica Estimulación del cerebro Microslices como<em&gt; In vitro</em&gt; Modelo de Carrera

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

DTI of the Visual Pathway - White Matter Tracts and Cerebral Lesions

DTI is a technique that identifies white matter tracts (WMT) non-invasively in healthy and non-healthy patients using diffusion measurements. Similar to visual pathways (VP), WMT are not visible with classical MRI or intra-operatively with microscope. DTI will help neurosurgeons to prevent destruction of the VP while removing lesions adjacent to this WMT. We have performed DTI on fifty patients before and after surgery between March 2012 to January 2014. To navigate we used a 3DT1-weighted sequence. Additionally, we performed a T2-weighted and DTI-sequences. The parameters used were, FOV: 200 x 200 mm, slice thickness: 2 mm, and acquisition matrix: 96 x 96 yielding nearly isotropic voxels of 2 x 2 x 2 mm. Axial MRI was carried out using a 32 gradient direction and one b0-image. We used Echo-Planar-Imaging (EPI) and ASSET parallel imaging with an acceleration factor of 2 and b-value of 800 s/mm&#178;. The scanning time was less than 9 min.
The DTI-data obtained were processed using a FDA approved surgical navigation system program which uses a straightforward fiber-tracking approach known as fiber assignment by continuous tracking (FACT). This is based on the propagation of lines between regions of interest (ROI) which is defined by a physician. A maximum angle of 50, FA start value of 0.10 and ADC stop value of 0.20 mm&#178;/s were the parameters used for tractography.
There are some limitations to this technique. The limited acquisition time frame enforces trade-offs in the image quality. Another important point not to be neglected is the brain shift during surgery. As for the latter intra-operative MRI might be helpful. Furthermore the risk of false positive or false negative tracts needs to be taken into account which might compromise the final results.

Diffusion tensor imaging (DTI) was performed to try to depict the major parts of the visual pathway. The goal was to use an FDA approved standard commercial workstation which could be used for everyday routine in order to try to reduce postoperative damage of the visual pathway in the patients.

DTI de la vía visual - la sustancia blanca Tracts y lesiones cerebrales

DTI is a technique that identifies white matter tracts (WMT) non-invasively in healthy and non-healthy patients using diffusion measurements. Similar to ...

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

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

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

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

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

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 ...

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic cerebrovascular accidents account for 80% of all strokes. A common cause of IR injury is the rapid inflow of fluids following an acute/chronic occlusion of blood, nutrients, oxygen to the tissue triggering the formation of free radicals.
Ischemic stroke is followed by blood-brain barrier (BBB) dysfunction and vasogenic brain edema. Structurally, tight junctions (TJs) between the endothelial cells play an important role in maintaining the integrity of the blood-brain barrier (BBB). IR injury is an early secondary injury leading to a non-specific, inflammatory response. Oxidative and metabolic stress following inflammation triggers secondary brain damage including BBB permeability and disruption of tight junction (TJ) integrity.
Our protocol presents an in vitro example of oxygen-glucose deprivation and reoxygenation (OGD-R) on rat brain endothelial cell TJ integrity and stress fiber formation. Currently, several experimental in vivo models are used to study the effects of IR injury; however they have several limitations, such as the technical challenges in performing surgeries, gene dependent molecular influences and difficulty in studying mechanistic relationships. However, in vitro models may aid in overcoming many of those limitations. The presented protocol can be used to study the various molecular mechanisms and mechanistic relationships to provide potential therapeutic strategies. However, the results of in vitro studies may differ from standard in vivo studies and should be interpreted with caution.

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is associated with a high rate of morbidity and mortality. The goal of the in vitro model of oxygen-glucose deprivation and reoxygenation (OGD-R) described here is to assess the effects of ischemia reperfusion injury on a variety of cells, particularly in blood-brain barrier (BBB) endothelial cells.

Oxígeno-Glucosa Privación y Reoxigenación como<em&gt; In Vitro</em&gt; La isquemia-reperfusión modelo para el estudio de la disfunción barrera hematoencefálica

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic ...

An Anesthesia, Surgery, and Harvest Method for the Evaluation of Transpedicular Screws Using an In Vivo Porcine Lumbar Spine Model

Pedicle screw fixation is the gold standard for the treatment of spinal diseases. However, many studies have reported the issue of loosening pedicle screws after spinal surgery, which is a serious concern. To address this problem, diverse types of pedicle screws have been examined to identify those with good fixation strength and osseointegration in spine bone. The porcine spine is a good alternative for the human spine in the evaluation of pedicle screws due to the anatomical size, mechanical characteristics, and cost. Although several studies have reported that pedicle screws are efficient in the porcine model, no study has described detailed protocols for the evaluation of a pedicle screw using the porcine model. Here, we describe a detailed method for evaluating transpedicular screws using an in vivo porcine lumbar spine model. The technical details for anesthesia, spine surgery, and harvest provided here will facilitate with the evaluation of the transpedicular screw fixation model.

An Anesthesia, Surgery, and Harvest Method for the Evaluation of Transpedicular Screws Using an In Vivo Porcine Lumbar Spine Model

Here, we introduce a method to evaluate transpedicular screws by using an in vivo porcine lumbar spine model.

Anestesia, Cirugía y Método de Cosecha para la Evaluación de Tornillos Transpediculares Usando un<em&gt; In Vivo</em&gt; Modelo de columna lumbar porcina

Pedicle screw fixation is the gold standard for the treatment of spinal diseases. However, many studies have reported the issue of loosening pedicle ...