Name: photothrombosis-induced focal ischemia as a model of spinal cord injury in mice
Uploaded: 2015-07-16T15:00:00
Description: Watch this Scientific Journal Video about Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice at JoVE.com

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...

<ol>
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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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	<tbody>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">C57Bl6/J</td>
			<td>Jackson lab</td>
			<td>664</td>
			<td>22-25g</td>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Artificial tears ointment&#160;</td>
			<td>Rugby</td>
			<td>NDC-0536-6550-91</td>
			<td>83% white petrolatum</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol</td>
			<td>Decon labs.Inc</td>
			<td>2716</td>
			<td>70% ethanol for disinfection</td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Standard scalpel</td>
			<td>Fine Science Tool</td>
			<td>10003-12</td>
			<td>for removing muscle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scalpel blade</td>
			<td>Feather</td>
			<td>2976</td>
			<td>No. 10</td>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forceps (straight)</td>
			<td>Fine Science Tool</td>
			<td>11151-10</td>
			<td>for holding tissue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Needle holder&#160;</td>
			<td>Fine Science Tool</td>
			<td>12002-12</td>
			<td>for suturing</td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Perma-hand silk</td>
			<td>Ethicon</td>
			<td>683G</td>
			<td>Size = 4-0 (for skin)</td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heating pad</td>
			<td>Fine Science Tool</td>
			<td>21052-00</td>
			<td>to prevent hypothermia</td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">X-Y gliding stage&#160;</td>
			<td>Amscope</td>
			<td>GT100</td>
			<td>for positioning the animal under microscope&#160;&#160;</td>
		</tr>
	</tbody>
</table>

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) ...

Research

JoVE Journal

Medicine

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

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

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

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

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

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

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

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

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

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

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 ...