Esta labor fue apoyada por los Institutos Nacionales de Salud [de Grant no. R01NS069726] y la Asociación Americana del Corazón Grant en Cooperación Financiera No Reembolsable [subvención no. 13GRNT17020004] para SD.

Nota: Los ratones (C57BL / 6J, macho) edad 10 - 12 semanas fueron usados ​​en este estudio. Todos los procedimientos se realizaron de conformidad con la Guía del NIH para el Cuidado y Uso de Animales de Laboratorio y fueron aprobados por la Universidad de Missouri Institucional Cuidado de Animales y el empleo Comisión (IACUC). 1. Pre-Op <ol><li> El día antes de la cirugía autoclave y esterilizar todos los instrumentos quirúrgicos. Envolver los instrumentos y autoclave a 121 ° C a 15 psi durante 30 min seguido por 30 min de secado (121 ° C, 15 psi, 30/30 ciclo). Coloque los instrumentos en un ambiente limpio y estéril hasta su uso posterior. </li><li> Prepare una solución fresca Rosa de Bengala (RB) (20 mg / ml en solución salina estéril) cada vez que antes de la cirugía. Para disolver completamente RB, vórtice del tubo y luego seguido de tratamiento con ultrasonidos durante 5 min a 50/60 Hz con potencia de salida de 19 W. Envuelva el tubo en papel de aluminio y protegerlo de la luz hasta su uso aún más durante surgery. </li><li> Preparar una mezcla de ketamina / xilazina en solución salina estéril. Añadir 125 l de xilazina (concentración al campo: 20 mg / ml) y 325 l de ketmaine (concentración al campo: 100 mg / ml) y 550 l de solución salina estéril para hacer un volumen final de 1 ml de mezcla anestésica. </li><li> Pre-calentar la almohadilla térmica homeotérmico. </li><li> Pre-calentar la lámpara de haluro metálico (fuente de luz para microscopio FN1 epi-fluorescencia) durante 30 minutos para estabilizar la potencia de la lámpara. </li><li> Ajuste del tamaño de la región iluminada a un diámetro de 1 mm utilizando objetivo de 10X y una retícula mediante el ajuste del diafragma de campo en el FN1 vertical microscopio epi-fluorescencia. </li></ol> 2. Procedimiento Quirúrgico <ol><li> Anestesiar al ratón con una dosis de ketamina (130 mg / kg B. peso.) Y xilazina (10 mg / kg B. peso.). Basado en el cóctel de 1,3, 4 l por g de ratón B. peso. Se necesitan. Esterilizar el sitio de la inyección con un algodón con alcohol y administrar los anestésicos a través de la intra-peritoneal ruta (IP). Tenga cuidado de no inyectar los anestésicos en los vasos sanguíneos o el músculo como esto retrasaría la inducción y la recuperación del animal. </li><li> Aplique un ungüento de lágrimas artificiales para los dos ojos del ratón para evitar que se seque y coloque el animal sobre el cojín eléctrico para evitar la hipotermia. </li><li> Preparación de los animales <ol><li> Compruebe el animal para la anestesia quirúrgica nivel adecuado mediante el uso de la respuesta pizca dedo del pie. </li><li> Una vez que el animal ha alcanzado el nivel quirúrgico de anestesia, cortar el pelo en la superficie dorsal alrededor de la línea media del animal utilizando un cortador de pelo eléctrico. Frote el sitio quirúrgico con etanol al 70%, seguido por la solución de betadine tres veces. Cubra el sitio con un paño quirúrgico estéril hasta el próximo paso. </li></ol></li><li> Procedimiento quirúrgico para diluir el hueso para exponer la médula espinal <ol><li> Coloque el ratón en una posición de decúbito prono sobre la almohadilla de calefacción homeotermos en la plataforma quirúrgica (Figura 1A). Asegura adecuadamente el ratónpostura mediante una abrazadera hocico para mantener una región de cuello alargado (Figura 1A, B). </li><li> Hacer una incisión (aproximadamente 1 cm de longitud), utilizando tijeras quirúrgicas a lo largo de la línea media dorsal se extiende desde la T9 vértebras torácicas a T12. Separar la piel para exponer el área quirúrgica. </li><li> El uso de un bisturí, borrar cuidadosamente el músculo para exponer las espinas dorsales en T9 - T12 vértebras. Deje de sangrado en cada paso aplicando una presión suave con un hisopo de algodón estéril. T10 separado - T12 vértebras desde el músculo circundante y asegurarlas mediante una abrazadera vertebral para estabilizar y evitar cualquier movimiento (Figura 1A, B). </li><li> Usando un taladro de alta velocidad con el hueso pulido broca, con cuidado y suavidad fina la superficie dorsal de la vértebra T10 o T11 para visualizar la vena vertebral posterior y otras embarcaciones pequeñas en la superficie dorsal de la SC (Figura 1C). </li><li> Para evitar el daño térmico debido al calor generado durante el procedimiento de adelgazamiento,aplicar una corriente suave y constante de solución salina normal junto con succión constante para eliminar los escombros. </li><li> Usando un bisturí suavizar cuidadosamente la superficie del hueso hasta que el vaso principal es claramente visible. Tenga cuidado de no dañar la médula espinal durante este proceso. </li><li> Una vez que el vaso sanguíneo se visualiza, RB administrar a una dosis de 30 mg / kg (peso corporal) a través de ruta seno retro-orbital usando una jeringa de insulina. </li><li> Medir el flujo sanguíneo utilizando un medidor de flujo Doppler láser después de 3 min después de la inyección RB si es necesario (Figura 2A, B). Mantener la asepsia durante todo el procedimiento. </li></ol></li></ol> 3. Inducción de PT <ol><li> Colocar el animal en una etapa ajustable posición XY en un Lab-Jack que puede ajustar la altura. Ajuste la posición del ratón por lo que la región expuesta de la médula espinal T11 está directamente bajo el objetivo de 10x del microscopio FN1 epi-fluorescencia (Figura 3A). </li><li> Ajuste el poder de la lifuente de lucha en el 12% e irradiar la región T11 con un diámetro de 0,75 mm en el medio de adelgazada de la médula espinal (Nota: esta región incluye la vena posterior de la médula y otros capilares) con una luz verde (longitud de onda 540-580 nm, que es logrado por el cubo de filtro en el microscopio) a través del objetivo de 10X para 2 min. Tomar imágenes al principio y al final de la irradiación (Figura 3B, C) ​​y registrar el tiempo del experimento en este punto. </li><li> Medir el flujo sanguíneo de nuevo por 10 min si es necesario mediante la colocación de la sonda Doppler láser a la misma posición por encima de la médula espinal como en 2.4.8 (Figura 2A, B). </li><li> Después de la irradiación de verificación para cualquier hemorragia y si ninguno encontrado procede a la sutura del animal. Suturar la fascia superficial junto con los músculos en ambos lados de la médula espinal utilizando una sutura absorbible o tamaño sutura de seda 4-0. Tenga cuidado de no dañar el SC expuesto. Suturar la piel con sutura de seda 4-0. Aplicar Betadine o yodoa los bordes de la piel después de la sutura. </li></ol> 4. Después de la cirugía Cuidado <ol><li> Después de sutura, coloque el animal sobre el cojín eléctrico para la recuperación. Después de la recuperación comprobar los animales en busca de signos de déficit neurológico al observar el movimiento de ambas extremidades traseras. No dejar al animal sin vigilancia hasta que se haya recuperado el conocimiento suficiente para mantener decúbito esternal. </li><li> La transferencia de los animales a la jaula. No devolver el animal que ha sido sometido a cirugía para la compañía de otros animales hasta que se recupere totalmente. </li><li> Compruebe los animales a intervalos regulares. En caso de déficits neurológicos severos, proporcionar atención adecuada como la evacuación de la vejiga, la administración de analgésicos (buprenorfina, 0,05-0,1 mg / kg). Compruebe si hay deshidratación y administrar por vía subcutánea salina normal en caso grave. Por lo general, la buprenorfina (0,1 mg / kg) se administró después de la sutura para aliviar el dolor en el sitio quirúrgico. </li><li> Si los animales no son inmediatamentesacrificados después de la cirugía, pondremos dieta de alto contenido de agua en el suelo de la jaula, por lo que los animales pueden llegar a la comida con facilidad. </li></ol> 5. transcardial de perfusión, la tinción de Nissl y inmunotinción <ol><li> Transcardially perfundir el animal como se describió anteriormente 17-20. <ol><li> Anestesiar al animal como se describió anteriormente en el protocolo y transcardially perfundir con solución salina tampón fosfato (PBS, pH 7,4), seguido de enfriado en hielo 4% de paraformaldehído (PFA) en PBS. </li><li> Después de la perfusión, retire la médula espinal (SC) y post-arreglarlo en el 4% PFA en PBS a 4 ° CO / N. Transferir el SC fijo en PBS con 30% de sacarosa y consérvelo para 2 - 3 días hasta que se hunde hasta el fondo del tubo. </li><li> El uso de un criostato cortar la médula espinal en 30 micras de espesor secciones y colocarlos en serie en un recubiertos de gelatina portaobjetos de vidrio o en una placa de 48 pocillos con 0,01 M PBS. </li></ol></li><li> Tinción de Nissl: Para inspeccionar el daño causado por PT realizar una Nissl staining en las secciones de la médula espinal como anteriormente descrito 17-20. <ol><li> Brevemente, recoja cada quinto trozo de la médula espinal en los portaobjetos de vidrio y se tiñen con 0.25% cresil violeta. Tomar imágenes de las secciones teñidas (Figura 4). </li></ol></li><li> La inmunotinción: Como se ha descrito anteriormente utilizando un método sección 17,18,20 flotante. <ol><li> Brevemente, manchar las secciones de la médula espinal mediante la incubación de O / N a 4 ° C con conejo anti-glia proteína fibrilar ácida (GFAP) anticuerpo policlonal (1: 300), anticuerpo de conejo anti-NeuN (1: 300), y anti-conejo Iba1 anticuerpo (1: 500) seguido de burro anti conejo Alexa 568-conjugado IgG (1: 400) anticuerpos secundarios durante 4 horas a RT. Tome imágenes con un microscopio de fluorescencia (Figura 5). </li></ol></li></ol>

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The authors have nothing to disclose.

En este estudio, hemos descrito un modelo photothrombotic de SC isquemia. Debido a los avances en la ingeniería genética no ha habido un aumento en los ratones transgénicos disponibles en el mercado que ha hecho posible el estudio del impacto de los genes específicos implicados en la fisiopatología isquémica en las CEC. El objetivo del estudio fue desarrollar un modelo reproducible de ratón de la isquemia de la médula espinal. Aquí hemos adaptado un modelo PT cortical para inducir SCI en ratone...

Lesión medular traumática (SCI) es una condición clínica devastadora que afecta a las funciones sensoriomotoras y autonómicas de la SC. Los pacientes que sobreviven SCI a menudo se dejan con paraplejia que afecta significativamente sus actividades y la calidad de vida 1 diarias debilitante. Modelos experimentales SCI han sido una herramienta indispensable en la investigación científica para comprender la fisiopatología de la lesión medular y los procesos de reparación neuronal asociados. Estos modelos también se han utilizado para probar la eficacia preclínica de varias intervenciones neuroprotectoras experimentales que tienen por objeto la recuperación funcional. Actualmente, la mayoría de los modelos de SCI en la práctica emplear el uso de la fuerza física contundente para interrumpir mecánicamente y lesionar a la SC. Estos métodos incluyen contusión, compresión, dislocación y transección del SC 2. Se ha sugerido que después de la lesión mecánica primaria una lesión secundaria en forma de conjuntos de isquemia en lesionada en el SC<html> 3,4. La etiología de la isquemia secundaria incluye una amplia degeneración de los tejidos, la hemorragia parenquimatosa y, a veces por la obstrucción de los vasos sanguíneos por edema tisular 5-7. Como resultado de la lesión secundaria de la integridad de SC se ve afectado aún más, las neuronas y las células gliales son severamente deteriorados en la función y viabilidad y sufren apoptosis que conduce a la del infarto de crecimiento durante la fase crónica de la lesión, de forma análoga al crecimiento de la penumbra isquémica después del accidente cerebrovascular 8,9. Varios mecanismos como excitotoxicidad, la producción de radicales libres y la inflamación se han notificado a ser responsable de la muerte celular isquémica tras SCI 10,11. Además, SC isquemia es una complicación grave de la cirugía de reparación de aneurisma de aorta toraco-abdominales que a menudo conducen a la paraplejia en los pacientes 12,13. A pesar de tal impacto clínico de alta muy pocos modelos de isquemia de la médula espinal con alta reproducibilidad están disponibles actualmente. nt &quot;&gt; Photothrombosis (PT) es un método comúnmente utilizado para la inducción de la isquemia focal en el cerebro 14-20. La técnica es bastante no invasiva, altamente reproducible y produce una lesión isquémica focal precisa en el área expuesta del cerebro 17 -21. Esto se logra mediante la administración sistémica de colorantes fotoactivos como Rosa de Bengala (RB) 16-20,22 o eritrosina B 23 seguido de irradiación localizada de vasos sanguíneos con fuente de luz apropiada. fotoactivación del colorante hace que la generación de radicales libres que perturbar la integridad del endotelio vascular liso, y hacen que las plaquetas se acumulen, que posteriormente se forma un trombo. La obstrucción del flujo sanguíneo por los resultados de trombos en un infarto en la región suministrada por el recipiente 24. Debido a la facilidad de control en el intensidad y duración de la irradiación Este procedimiento produce un infarto muy uniforme y reproducible. Además, este método puede ser empleado para inducir una infarct en varios lugares anatómicos permitiendo espacial (por ejemplo, la materia gris vs. materia blanca) comprensión de los efectos de la isquemia. El objetivo del presente estudio es desarrollar un modelo fácil y altamente reproducible de SC isquemia en ratones. Hemos descrito el procedimiento de un modelo de PT de SC isquemia en ratones. Los resultados de la histología y la inmunotinción demostraron que PT puede inducir eficazmente miocardio SC, la pérdida neuronal y gliosis reactiva.

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			<td height="21" style="height:21px;width:216px;">&#160;Rose Bengal</td>
			<td style="width:233px;">Sigma-Aldrich</td>
			<td style="width:180px;">330000</td>
			<td style="width:421px;">20 mg/ml in sterile saline</td>
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			<td height="21" style="height:21px;">C57Bl6/J</td>
			<td>Jackson lab</td>
			<td>664</td>
			<td>22-25g</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Ketamine&#160;</td>
			<td>VEDCO</td>
			<td>NDC-50989-996-06</td>
			<td>100 mg/ml</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Xylazine&#160;</td>
			<td>VEDCO</td>
			<td>NDC-50989-234-11</td>
			<td>100 mg/ml</td>
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			<td height="21" style="height:21px;">Betadine solution</td>
			<td>Purdue</td>
			<td>NDC-67618-150-01</td>
			<td>10% povidone iodine topical solution</td>
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			<td height="21" style="height:21px;">Normal saline</td>
			<td>Abott Laboratories</td>
			<td>04930-04-10</td>
			<td>For diluting RB, anaesthesia and for preventing tissue from drying</td>
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			<td height="21" style="height:21px;">Artificial tears ointment&#160;</td>
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			<td>83% white petrolatum</td>
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			<td height="21" style="height:21px;">Ethanol</td>
			<td>Decon labs.Inc</td>
			<td>2716</td>
			<td>70% ethanol for disinfection</td>
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			<td height="21" style="height:21px;">Metal halide lamp</td>
			<td>EXFO, Canada</td>
			<td>X-Cite 120 PC&#160;</td>
			<td>Set power at 12%</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Spring scissors&#160;</td>
			<td>Fine Science Tool</td>
			<td>15000-10</td>
			<td>for minor dissection</td>
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			<td height="21" style="height:21px;">Scissors (angled to side)</td>
			<td>Fine Science Tool</td>
			<td>14063-011</td>
			<td>No. 3 handle</td>
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			<td height="21" style="height:21px;">Standard scalpel</td>
			<td>Fine Science Tool</td>
			<td>10003-12</td>
			<td>for removing muscle</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Scalpel blade</td>
			<td>Feather</td>
			<td>2976</td>
			<td>No. 10</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Forceps (curved)</td>
			<td>Fine Science Tool</td>
			<td>11150-10</td>
			<td>for holding tissue</td>
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			<td height="21" style="height:21px;">Forceps (straight)</td>
			<td>Fine Science Tool</td>
			<td>11151-10</td>
			<td>for holding tissue</td>
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			<td height="21" style="height:21px;">Needle holder&#160;</td>
			<td>Fine Science Tool</td>
			<td>12002-12</td>
			<td>for suturing</td>
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			<td height="21" style="height:21px;">Tissue adhesive glue</td>
			<td>3M Vetbond</td>
			<td>1469SB</td>
			<td>to adhere to edges of the cut skin</td>
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			<td height="21" style="height:21px;">Monofilament polypropylene&#160;</td>
			<td>USSC Sutures</td>
			<td>VP-521</td>
			<td>Size = 4-0 (for fascia)</td>
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			<td height="21" style="height:21px;">Perma-hand silk</td>
			<td>Ethicon</td>
			<td>683G</td>
			<td>Size = 4-0 (for skin)</td>
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			<td height="21" style="height:21px;">Micro drill</td>
			<td>Roboz Surgical Instrument Co. Inc.</td>
			<td>RS-6300</td>
			<td>with bone polishing drill bit</td>
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			<td height="21" style="height:21px;">Laser doppler flowmeter</td>
			<td>Moor Instruments</td>
			<td>moorVMS-LDF1</td>
			<td>for monitoring change in blood flow</td>
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			<td height="21" style="height:21px;">Heating pad</td>
			<td>Fine Science Tool</td>
			<td>21052-00</td>
			<td>to prevent hypothermia</td>
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			<td height="21" style="height:21px;">Lab-Jack</td>
			<td>Fisher scientific&#160;</td>
			<td>14-673-50</td>
			<td>4x4 in plate to adjust the height of the animal</td>
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			<td height="21" style="height:21px;">X-Y gliding stage&#160;</td>
			<td>Amscope</td>
			<td>GT100</td>
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photothrombosis-induced focal ischemia as a model of spinal cord injury in mice

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) below the site of injury. The secondary ischemia that develops following the initial mechanical insult is a serious complication of the SCI and severely impairs the function and viability of surviving neuronal and non-neuronal cells in the SC. In addition, ischemia is also responsible for the growth of lesion during chronic phase of injury and interferes with the cellular repair and healing processes. Thus there is a need to develop a spinal cord ischemia model for studying the mechanisms of ischemia-induced pathology. Focal ischemia induced by photothrombosis (PT) is a minimally invasive and very well established procedure used to investigate the pathology of ischemia-induced cell death in the brain. Here, we describe the use of PT to induce an ischemic lesion in the spinal cord of mice. Following retro-orbital sinus injection of Rose Bengal, the posterior spinal vein and other capillaries on the dorsal surface of SC were irradiated with a green light resulting in the formation of a thrombus and thus ischemia in the affected region. Results from histology and immunochemistry studies show that PT-induced ischemia caused spinal cord infarction, loss of neurons and reactive gliosis. Using this technique a highly reproducible and relatively easy model of SCI in mice can be achieved that would serve the purpose of scientific investigations into the mechanisms of ischemia induced cell death as well as the efficacy of neuroprotective drugs. This model will also allow exploration of the pathological changes that occur following SCI in live mice like axonal degeneration and regeneration, neuronal and astrocytic Ca2+ signaling using two-photon microscopy.

El objetivo de este estudio era producir isquemia de la médula espinal en ratones utilizando un modelo de PT. Después de la región deseada de hueso por encima de la médula espinal (T10 - T12) se diluyó, Rosa de Bengala se inyectó por vía seno retro-orbital, y la isquemia fue inducida por PT Figura 1A, B muestran el ratón posicionado en una medida quirúrgica. plataforma durante la cirugía. El ratón se mantiene en su lugar por una abrazadera hocico y dos vertebrado ajustable abrazaderas para es...

Watch this Scientific Journal Video about Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice at JoVE.com

Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice

Photothrombosis is a minimally invasive and highly reproducible procedure to induce focal ischemia in the spinal cord and serves as a model of spinal cord injury in mice.

Photothrombosis inducida por isquemia focal como un modelo de lesión de médula espinal en ratones

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) ...

photothrombosis-induced-focal-ischemia-as-model-spinal-cord-injury

Research

JoVE Journal

Medicine

13.1K vistas.  University of Missouri. Photothrombosis is a minimally invasive and highly reproducible procedure to induce focal ischemia in the spinal cord and serves as a model of spinal cord injury in mice. 

Video: Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice

Acute and Chronic Tactile Sensory Testing after Spinal Cord Injury in Rats

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory testing in rat SCI models is needed. However, until recently, no single testing approach had been validated for SCI so that standardized methods have not been implemented across labs. Additionally, available testing methods could not be implemented acutely or when severe motor impairments existed, preventing studies of the development of SCI-induced allodynia3. Here we present two validated sensory testing methods using von Frey Hair (VFH) monofilaments which quantify changes in tactile sensory thresholds after SCI4-5. One test is the well-established Up-Down test which demonstrates high sensitivity and specificity across different SCI severities when tested chronically5. The other test is a newly-developed dorsal VFH test that can be applied acutely after SCI when allodynia develops, prior to motor recovery4-5. Each VFH monofilament applies a calibrated force when touched to the skin of the hind paw until it bends. In the up-down method, alternating VFHs of higher or lower forces are used on the plantar L5 dermatome to delineate flexor withdrawal thresholds. Successively higher forces are applied until withdrawal occurs then lower force VFHs are used until withdrawal ceases. The tactile threshold reflects the force required to elicit withdrawal in 50% of the stimuli. For the new test, each VFH is applied to the dorsal L5 dermatome of the paw while the rat is supported by the examiner. The VFH stimulation occurs in ascending order of force until at least 2 of 3 applications at a given force produces paw withdrawal. Tactile sensory threshold is the lowest force to elicit withdrawal 66% of the time. Acclimation, testing and scoring procedures are described. Aberrant trials that require a retest and typical trials are defined. Animal use was approved by Ohio State University Animal Care and Use Committee.

We describe two tactile sensory testing methods for acute or chronic periods of spinal cord injury in rats. These validated procedures can detect the development and maintenance of allodynia-like sensations.

Aguda y crónica Prueba sensorial táctil después de la lesión de la médula espinal en ratas

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory ...

Investigación

Medicina

Controlled Cervical Laceration Injury in Mice

Use of genetically modified mice enhances our understanding of molecular mechanisms underlying several neurological disorders such as a spinal cord injury (SCI). Freehand manual control used to produce a laceration model of SCI creates inconsistent injuries often associated with a crush or contusion component and, therefore, a novel technique was developed. Our model of cervical laceration SCI has resolved inherent difficulties with the freehand method by incorporating 1) cervical vertebral stabilization by vertebral facet fixation, 2) enhanced spinal cord exposure, and 3) creation of a reproducible laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm in depth without associated contusion. Compared to the standard methods of creating a SCI laceration such as freehand use of a scalpel or scissors, our method has produced a consistent lesion. This method is useful for studies on axonal regeneration of corticospinal, rubrospinal, and dorsal ascending tracts.

A novel technique to create a reproducible in vivo model of cervical spinal cord laceration injury in the mouse is described. This technique is based on spine stabilization by fixation of the cervical facets and laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm.

Controlado lesiones laceración cervical en ratones

Use of genetically modified mice enhances our  understanding of molecular mechanisms underlying several neurological disorders  such as a spinal cord ...

A Contusion Model of Severe Spinal Cord Injury in Rats

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is described to obtain a consistent model of severe SCI. Use of a stereotactic frame and computer controlled impactor allows for creation of reproducible injury. Hypothermia and urinary tract infection pose significant challenges in the post-operative period. Careful monitoring of animals with daily weight recording and bladder expression allows for early detection of post-operative complications. The functional results of this contusion model are equivalent to transection models. The contusion model can be utilized to evaluate the efficacy of both neuroprotective and neuroregenerative approaches.

A contusion model of severe spinal cord injury is described. Detailed pre-operative, operative and post-operative steps are described to obtain a consistent model.

Un modelo Contusión de la lesión de la médula espinal severa en ratas

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is ...

Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often unreported mortality rates that may confound analyses. Bilateral renal pedicle clamping is commonly used to induce IR-AKI, but differences between effective clamp pressures and/or renal responses to ischemia between kidneys often lead to more variable results. In addition, shorter clamp times are known to induce more variable tubular injury, and while mice undergoing bilateral injury with longer clamp times develop more consistent tubular injury, they often die within the first 3 days after injury due to severe renal insufficiency. To improve post-injury survival and obtain more consistent and predictable results, we have developed two models of unilateral ischemia-reperfusion injury followed by contralateral nephrectomy. Both surgeries are performed using a dorsal approach, reducing surgical stress resulting from ventral laparotomy, commonly used for mouse IR-AKI surgeries. For induction of moderate injury BALB/c mice undergo unilateral clamping of the renal pedicle for 26 min and also undergo simultaneous contralateral nephrectomy. Using this approach, 50-60% of mice develop moderate AKI 24 hr after injury but 90-100% of mice survive. To induce more severe AKI, BALB/c mice undergo renal pedicle clamping for 30 min followed by contralateral nephrectomy 8 days after injury. This allows functional assessment of renal recovery after injury with 90-100% survival. Early post-injury tubular damage as well as post injury fibrosis are highly consistent using this model.

We describe models of moderate and severe ischemia-reperfusion-induced kidney injury in which the mice undergo unilateral renal pedicle clamping followed by simultaneous or delayed contralateral nephrectomy, respectively. These models consistently give rise to renal dysfunction and post-injury fibrosis, but injury severity and survival are dependent on mouse background, age and surgical equipment.

La isquemia-reperfusión modelo de lesión renal aguda y post lesión fibrosis en ratones

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often ...

Reproducable Paraplegia by Thoracic Aortic Occlusion in a Murine Model of Spinal Cord Ischemia-reperfusion

Background 
Lower extremity paralysis continues to complicate aortic interventions. The lack of understanding of the underlying pathology has hindered advancements to decrease the occurrence this injury.&#160;The current model demonstrates reproducible lower&#160;extremity paralysis following thoracic aortic occlusion.
Methods 
Adult male C57BL6 mice were anesthetized with isoflurane. Through a cervicosternal incision the aorta was exposed. The descending thoracic aorta and left subclavian arteries were identified without entrance into pleural space. Skeletonization of these arteries was followed by immediate closure (Sham) or occlusion for 4 min (moderate ischemia) or 8 min (prolonged ischemia). The sternotomy and skin were closed and the mouse was transferred to warming bed for recovery.&#160; Following recovery, functional analysis was obtained at 12 hr intervals until 48 hr.
Results 
Mice that underwent sham surgery showed no observable hind&#160;limb deficit. Mice subjected to moderate ischemia for 4 min had minimal functional deficit at 12 hr followed by progression to complete paralysis at 48 hr. Mice subjected to prolonged ischemia had an immediate paralysis with no observable hind-limb movement at any point in the postoperative period. There was no observed intraoperative or post&#160;operative mortality.
Conclusion 
Reproducible lower extremity paralysis whether immediate or delayed can be achieved in a murine model. Additionally, by using a median sternotomy and careful dissection, high survival rates, and reproducibility can be achieved.

The lack of mechanistic understanding of spinal cord ischemia-reperfusion injury has hindered further adjuncts to prevent paraplegia following high risk aortic operations. Thus, the development of animal models is imperative. This manuscript demonstrates reproducible lower extremity paralysis following thoracic aortic occlusion in a murine model.

Paraplejia reproducible por la oclusión de la aorta torácica en un modelo murino de la médula espinal por isquemia-reperfusión

Background
Lower extremity paralysis continues to complicate aortic interventions. The lack of understanding of the underlying pathology has hindered ...

Controlled Cortical Impact Model for Traumatic Brain Injury

Every year over a million Americans suffer a traumatic brain injury (TBI). Combined with the incidence of TBIs worldwide, the physical, emotional, social, and economical effects are staggering. Therefore, further research into the effects of TBI and effective treatments is necessary. The controlled cortical impact (CCI) model induces traumatic brain injuries ranging from mild to severe. This method uses a rigid impactor to deliver mechanical energy to an intact dura exposed following a craniectomy. Impact is made under precise parameters at a set velocity to achieve a pre-determined deformation depth. Although other TBI models, such as weight drop and fluid percussion, exist, CCI is more accurate, easier to control, and most importantly, produces traumatic brain injuries similar to those seen in humans. However, no TBI model is currently able to reproduce pathological changes identical to those seen in human patients. The CCI model allows investigation into the short-term and long-term effects of TBI, such as neuronal death, memory deficits, and cerebral edema, as well as potential therapeutic treatments for TBI.

Traumatic brain injuries (TBIs) remain a serious health problem. Using the controlled cortical impact surgery model, research on the effects of TBI and possible treatment methods may be performed.

Controlado Modelo de Impacto cortical de la lesión cerebral traumática

Every year over a million Americans suffer a traumatic brain injury (TBI).  Combined with the incidence of TBIs worldwide, the physical, emotional, ...

A Radio-telemetric System to Monitor Cardiovascular Function in Rats with Spinal Cord Transection and Embryonic Neural Stem Cell Grafts

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter connected to a radio transmitter into the femoral artery of rats that underwent a T4 spinal cord transection with or without grafting of embryonic brainstem-derived neural stem cells expressing green fluorescent protein. Compared to other methods such as cannula insertion or tail-cuff, telemetry is advantageous to continuously monitor blood pressure and heart rate in freely moving animals. It is also capable of long term multiple data acquisitions. In spinal cord injured rats, basal cardiovascular data under unrestrained condition and autonomic dysreflexia in response to colorectal distension were successfully recorded. In addition, cardiovascular parameters before and after SCI can be compared in the same rat if a transmitter is implanted before a spinal cord transection. One limitation of the described telemetry procedure is that implantation in the femoral artery may influence the blood supply to the ipsilateral hindlimb.

We present a protocol for using a radio-telemetric system to record cardiovascular parameters in T4 spinal cord transected rats eight weeks after embryonic brainstem neural stem cell grafting into the lesion site. Telemetry is an advanced technique to accurately evaluate cardiovascular function in conscious freely moving spinal cord injured rats.

Un sistema de radio-telemetría para monitorizar la función cardiovascular en ratas con la médula espinal y transección Embryonic Stem Neural injertos de células

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter ...

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can be used both to investigate the biological responses to injury and to test potential therapies. Contusion or compression injury delivered to the surgically exposed spinal cord are the most widely used models of the pathology. In this report the experimental contusion is performed by using the Infinite Horizon (IH) Impactor device, which allows the creation of a reproducible injury animal model through definition of specific injury parameters. Stem cell transplantation is commonly considered a potentially useful strategy for curing this debilitating condition. Numerous studies have evaluated the effects of transplanting a variety of stem cells. Here we demonstrate an adapted method for spinal cord injury followed by tail vein injection of cells in CD1 mice. In short, we provide procedures for: i) cell labeling with a vital tracer, ii) pre-operative care of mice, iii) execution of a contusive spinal cord injury, and iv) intravenous administration of post mortem neural precursors. This contusion model can be utilized to evaluate the efficacy and safety of stem cell transplantation in a regenerative medicine approach.

Spinal cord injury is a traumatic condition that causes severe morbidity and high mortality. In this work we describe in detail a contusion model of spinal cord injury in mice followed by a transplantation of neural stem cells.

Neural Trasplante de células madre en Experimental contuso Modelo de Lesión de la Médula Espinal

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can ...

Calibrated Forceps Model of Spinal Cord Compression Injury

Compression injuries of the murine spinal cord are valuable animal models for the study of spinal cord injury (SCI) and spinal regenerative therapy. The calibrated forceps model of compression injury is a convenient, low cost, and very reproducible animal model for SCI. We used a pair of modified forceps in accordance with the method published by Plemel et al. (2008) to laterally compress the spinal cord to a distance of 0.35 mm. In this video, we will demonstrate a dorsal laminectomy to expose the spinal cord, followed by compression of the spinal cord with the modified forceps. In the video, we will also address issues related to the care of paraplegic laboratory animals. This injury model produces mice that exhibit impairment in sensation, as well as impaired hindlimb locomotor function. Furthermore, this method of injury produces consistent aberrations in the pathology of the SCI, as determined by immunohistochemical methods. After watching this video, viewers should be able to determine the necessary supplies and methods for producing SCI of various severities in the mouse for studies on SCI and/or treatments designed to mitigate impairment after injury.

Spinal cord injury models should be highly reproducible. We demonstrate that the calibrated forceps compression model of spinal cord injury is an easy to use surgical method for generating reproducible injuries to the murine spinal cord.

Calibrado Pinzas Modelo de Lesión Medular Compresión

Compression injuries of the murine spinal cord are valuable animal models for the study of spinal cord injury (SCI) and spinal regenerative therapy. The ...

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

Un ratón neonatal lesión de médula espinal de compresión Modelo

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...