The work was supported by grants from NIH/NINDS (NS054883), Craig H. Neilsen Foundation (280072), and the Veterans Administration and Canadian Spinal Research Organization. We thank the Rat Resource and Research Center, University of Missouri, Columbia, Missouri, for providing GFP rats.

Todos los animales protocolos fueron aprobados por el Cuidado de Animales y el empleo Comisión Institucional (IACUC). Directrices del NIH para el cuidado de animales de laboratorio y seguridad se siguen estrictamente. Los animales con los procedimientos quirúrgicos fueron tratados adecuadamente para minimizar el dolor y el malestar. 1. Médula Espinal Curgery e injerto celular <ol><li> Instrumentos quirúrgicos autoclave antes de todas las cirugías. Utilice Hot bolas Esterilizador (Herramientas de Bellas Ciencia) para eliminar los agentes patógenos y contaminantes microbianos de instrumentos entre los procedimientos en diferentes animales. Utilice guantes estériles quirúrgicos, batas y cortinas durante la cirugía. Emplear técnica quirúrgica aséptica para cada procedimiento quirúrgico. </li><li> Anestesie hembra Fischer 344 ratas con una combinación (2 ml / kg) de ketamina (25 mg / ml), xilazina (1,3 mg / ml), y acepromacina (0,25 mg / ml) administrados a través de inyección intraperitoneal (ip). </li><li> Afeitarse la zona de la espalda y limpiar la piel en varias ocasiones con Betadine y etanol.</li><li> Cortar la piel con una cuchilla # 10 y sin rodeos disecar los músculos capas. Utilice un bisturí para aislar las vértebras, exponer la vértebra T3, y realizar una laminectomía dorsal usando una punta fina gubia. </li><li> Incisión en la duramadre longitudinalmente y seccionar la médula espinal a nivel de T4 utilizando una combinación de iridectomía tijeras y microaspiración para crear un espacio de rostrocaudal aproximadamente 1 mm entre los muñones de la médula espinal. </li><li> Espere alrededor de 1 - 2 min hasta que el sangrado se detenga y luego totalmente los músculos de sutura con 3-0 Vycril y cerrar la piel con grapas para heridas. </li><li> Inyectar con lactato de Ringer (5 ml), buprenorfina (0,035 mg / kg) y ampicilina (33 mg / kg) por vía subcutánea inmediatamente después de la cirugía y mantener ratas en una incubadora caliente hasta que despierta. </li><li> Se inyecta la solución Ampicilina una vez al día hasta 10 días, y la buprenorfina dos veces al día durante 3 días o hasta que aparezcan signos de dolor y angustia desaparecen de Ringer y. No devuelva un animal a la compañía de otros animales hasta fully recuperó. </li><li> Vaciar manualmente la vejiga dos veces al día durante aproximadamente dos semanas hasta que el establecimiento de la vejiga reflejo de vaciado, y luego vaciar la vejiga una vez al día a lo largo de la supervivencia si es necesario. </li><li> Dos semanas después, volver a anestesiar ratas SCI como se describe anteriormente y volver a exponer el sitio de la lesión de la médula espinal. Mantenga la duramadre cerrado para retener las células implantadas en el sitio de la lesión. </li><li> Inyectar 10 l de solución de células (3,5 × 10 5 l), obtenida de GFP ubicua E14 embriones de rata transgénica y embebidos en fibrinógeno y trombina 5,6, en el epicentro de la cavidad de la lesión y la interfaz rostral y caudal de lesión con inyección múltiple sitios, utilizando una micropipeta de vidrio desmenuzado con un diámetro interior de 40 m, conectada a una jeringa Hamilton. </li><li> Suturar las capas musculares y cerrar la piel con grapas para heridas. </li><li> Lleve a cabo después de la inyección de la cirugía y el cuidado de la vejiga, como se describe en los pasos 1.6 a 1.8. </li><li> Una semana estarla perfusión tanto, se inyectan 0,5% Fluorogold (FG, 0,4 ml en agua destilada) por vía intraperitoneal a la etiqueta retrógradamente las neuronas preganglionares simpáticas en la médula espinal 7. </li></ol> 2. Transmisor Implantación <ol><li> Remojar transmittersin solución de glutaraldehído al 2% (4 ml de 50% glutaraldhyde en 96 ml de agua destilada) durante al menos 1 - 2 horas (hasta 10 horas) a temperatura ambiente durante la esterilización. </li><li> Lávese a fondo con transmisores estéril al 0,9% de solución salina 3 veces y almacenarlos en solución salina hasta su uso (no más de 1 hora). </li><li> Ocho semanas después del trasplante de células E14 (10 semanas después de la lesión), reanesthetize las ratas que se sometieron a un SCI con o sin injerto celular y las ratas de control no tratados previamente. </li><li> Afeitar el área abdominal y extremidades posteriores. Limpie la piel con Betadine. Coloque la rata en la mesa de operaciones en posición supina. </li><li> Incisión en la piel en el abdomen y el muslo interno ventral en el lado derecho utilizando una cuchilla # 15. </li><li> Corte a través de los stejidos conectivos ubcutaneous para exponer el conjunto de vasos femorales y nervio utilizando una tijera pequeña. </li><li> Separar la arteria femoral de la vena y el nervio usando una pinza fina con puntas curvadas. </li><li> Ponga tres suturas de seda debajo de la arteria y hacer un nudo flojo en cada sutura. </li><li> Aplicar 0,1 ml de lidocaína (2%) a la superficie de la arteria para provocar la vasodilatación para su posterior cateterización. </li><li> Asegure el recipiente distalmente con un nudo de seda permanente y temporal de bloques proximalmente por el estiramiento de una sutura de seda floja. </li><li> La punción de la arteria mediante una aguja curvada de calibre 20 e inserte la punta del catéter telémetro (8 cm de largo) con una herramienta de inserción del catéter. </li><li> Inserte el catéter rostral hasta 4 cm que coloca a la punta en la aorta torácica. </li><li> Anclar el catéter dentro del vaso mediante la vinculación de tres suturas de seda alrededor de la arteria femoral. </li><li> Hacer un bolsillo subcutáneo a lo largo del flanco entre el borde caudal de la caja torácica y el tque la extensión más craneal de gama de la rodilla con unas tijeras romas. </li><li> Insertar el cuerpo del transmisor en el bolsillo y la sutura al tejido conectivo que rodea el transmisor para evitar el movimiento excesivo. </li><li> Suturar la piel con el número 6 de hilo de seda. </li><li> Inyectar con lactato de Ringer (5 ml), buprenorfina (0,035 mg / kg) y ampicilina (33 mg / kg) por vía subcutánea inmediatamente después de la cirugía y mantener ratas en una incubadora caliente hasta que despierta. </li></ol> 3. Basal presión arterial media (PAM) y la frecuencia cardíaca (FC) de grabación <ol><li> Ya en un día después de la implantación del transmisor, poner un solo animal en la plataforma de receptor y encienda el transmisor. Espere unos 10 - 15 minutos a habituar al animal y estabilizar los parámetros cardiovasculares. </li><li> Registro de PAM y FC de reposo, que se derivan de la presión arterial del pulso con un sistema de adquisición de datos computarizado durante al menos 1 h. Recopilar datos cada 5 segundos. </li><li> Monitorear la unaanimalesque eliminan continuamente y puntos de datos durante la ocurrencia de los espasmos visibles. Para cada animal, los puntos de datos promedio para obtener valores medios. </li></ol> 4. colorrectal Distensión inducida Disreflexia Autónoma <ol><li> Restringir ratas NSC injertados o de control SCI en una toalla suministrado con bolitas de comida en el interior del receptor transmisor. Por lo general, las ratas pueden permanecer cooperativamente durante el procedimiento. </li><li> Insertar un catéter con punta de globo de látex en el recto durante aproximadamente 2 cm y fijarlo a la cola con cinta 8. </li><li> Encienda el transmisor y esperar durante 10 - 15 min permitiendo que la presión de la sangre para volver al inicio del estudio previo a la inserción. </li><li> Inducir la distensión colorrectal mediante el inflado del globo lentamente durante 10 segundos con 1,4 ml de aire por 1 min, para generar una presión de aproximadamente 30 mmHg. </li><li> MAPA Grabar y HR 1 min antes, durante 1 min, y 1 min después de la distensión colorrectal; datos de ejemplo cada 3 s durante el procedimiento de 3 min. </li><li>Realizar 2 - 3 ensayos por animal con al menos 15 min intervalo de recuperación entre dos ensayos. </li><li> Animales de sobredosis (ip) con la combinación de anestesia dosis doble descrito anteriormente, si no más evaluación. Perfundir animales con solución salina seguido de paraformaldehído al 4%. </li><li> Para cada animal, la media de los valores antes y durante la distensión colorrectal, respectivamente; calcular la diferencia entre el valor inicial y el PAM y FC cambios inducidos por distensión-para cada ensayo; promedio durante los 2 - 3 ensayos para obtener los valores medios. </li></ol> 5. Transmisor Limpieza <ol><li> Retire el transmisor del cuerpo de un animal después de la anestesia, pero antes de la perfusión. Remojar inmediatamente en un vaso de precipitados lleno con agua destilada hasta que la limpieza; evitar el secado del dispositivo de telémetro. </li><li> Transferir el telémetro a la solución de limpieza enzimática 1% Terg-A-Zyme (10 g de agua / L) durante 24 horas a temperatura ambiente. </li><li> Regel la punta del catéter del transmisor con una aguja de punta roma de calibre 30conectado a la jeringa regel. </li><li> Secar cuidadosamente el transmisor usando un paño suave plegada y almacenarlo en la bandeja de plástico original. </li></ol>

<ol>
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Los autores no tienen nada que revelar.

Tradicionalmente, una cánula llena de líquido se inserta en la arteria y se conecta a un transductor de presión para registrar los parámetros cardiovasculares como una instantánea terminal en cada animal 11. Para supervisar continuamente el rendimiento cardiovascular durante mucho tiempo, los sistemas de radio-telemétrico se emplean en muchos laboratorios. Esta herramienta más refinada puede registrar la presión arterial en el consciente, moviéndose libremente animales. En comparación con los catét...

Disfunción cardiovascular se produce después de una lesión de la médula espinal (SCI) en niveles altos. Se manifiesta en la presión desordenada arterial y frecuencia cardíaca en reposo, hipotensión ortostática, hipotensión inducida por el ejercicio, y la DA se caracteriza por episodios de hipertensión y bradicardia baroreflex mediada en respuesta a estímulos sensoriales por debajo del nivel de la lesión 1,2. Estos síntomas interfieren con la vida diaria de los pacientes con lesión medular. Por lo tanto, es importante establecer herramientas eficaces para la investigación de cambios cardiovasculares en animales con SCI y los tratamientos experimentales. Para investigar la función cardiovascular en animales, varias técnicas se han utilizado para controlar la presión arterial y la frecuencia cardíaca. Los parámetros cardiovasculares centrales pueden ser registrados por inserción de la cánula y la telemetría, mientras que la cola no invasivos esposas se pueden emplear para medir la presión arterial periférica 3. En comparación con otros métodos, telemetría tiene la ventaja principal que permite la grabación continua en animales con libertad de movimientos y el seguimiento a largo plazo de la función cardiovascular 4. En modelos animales de SCI, los cambios en la presión de la sangre periférica después de la estimulación experimental no pueden ser lo suficientemente grande para ser detectado. En consecuencia, la técnica de monitorización cardiaca adecuada debe ser seleccionado para animales con SCI. En el presente estudio, un sistema de radio-telemetría se introdujo para controlar la función cardiovascular en ratas adultas después de la sección completa de la médula espinal. Las ratas recibieron injertos de rata singénico día embrionario 14 (E14) células madre neuronales del tronco cerebral derivado (BS-NSC) en el sitio de la lesión. Ratas con lesiones y no de trasplante e ingenuos, ratas ilesos sirvieron como controles. El procedimiento de la telemetría incluye la esterilización transmisor y la implantación (Figura 1), la grabación del parámetro cardiovascular basal, respuestas inducidas por distensión colorrectal, y la limpieza y transmisoralmacenamiento.

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		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:315px;">Table of Specific Materials/Equipment:</td>
			<td style="width:103px;"></td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="20">
			<td height="41" rowspan="2" style="height:41px;width:120px;">Reagents</td>
			<td rowspan="2" style="width:195px;">Company</td>
			<td rowspan="2" style="width:103px;">Catalogue</td>
			<td rowspan="2" style="width:164px;">Comment</td>
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		<tr height="21">
		</tr>
		<tr height="62">
			<td height="83" rowspan="2" style="height:83px;width:120px;">Fibrinogen (rat)</td>
			<td rowspan="2" style="width:195px;">Sigma</td>
			<td rowspan="2" style="width:103px;">F6755-25MG</td>
			<td rowspan="2" style="width:164px;">2 hr at 37oC to dissovle</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="59">
			<td height="80" rowspan="2" style="height:80px;width:120px;">Thrombin (rat)</td>
			<td rowspan="2" style="width:195px;">Sigma</td>
			<td rowspan="2" style="width:103px;">T5772-100UN</td>
			<td rowspan="2" style="width:164px;">Dissovle in 10 mM CaCl2</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:120px;">1% Terg-A-Zyme</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:103px;">Z273287</td>
			<td style="width:164px;">Enzymatic solution for telemeter cleaning</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:120px;">Fluorogold</td>
			<td style="width:195px;">Fluorochrome</td>
			<td style="width:103px;"></td>
			<td style="width:164px;">Dissovle in distilled water and avoid light</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:120px;">Telemeter&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; (PA-C40)</td>
			<td style="width:195px;">Data Sciences International</td>
			<td style="width:103px;"></td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:120px;">Telementric recording and analysis system</td>
			<td style="width:195px;">Data Sciences International</td>
			<td style="width:103px;"></td>
			<td style="width:164px;">Signal stimulator, Data Exchange Matrix, receivers, Ambient pressure reference monitor</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:120px;">Balloon-tipped catheter</td>
			<td style="width:195px;">Edward Lifesciences</td>
			<td style="width:103px;">111F7-P</td>
			<td style="width:164px;">For colorectal distension</td>
		</tr>
	</tbody>
</table>

a radio-telemetric system to monitor cardiovascular function in rats with spinal cord transection and embryonic neural stem cell grafts

Lesión de la médula espinal torácica o cervical alta (SCI) puede conducir a la disfunción cardiovascular. Para controlar los parámetros cardiovasculares, se implantó un catéter conectado a un transmisor de radio en la arteria femoral de ratas que se sometieron a una transección de la médula espinal T4 con o sin injerto de células madre neurales embrionarias del tallo cerebral derivados que expresan la proteína verde fluorescente. En comparación con otros métodos, como la inserción de la cánula o manguito de la cola, la telemetría es ventajoso para controlar continuamente la presión arterial y la frecuencia cardíaca en los animales se mueven libremente. También es capaz de múltiples adquisiciones de datos a largo plazo. En la médula espinal lesionada ratas, se registraron los datos con éxito cardiovasculares basales bajo condición desenfrenada y la DA en respuesta a la distensión colorrectal. Además, los parámetros cardiovasculares antes y después de SCI pueden compararse en la misma rata si un transmisor se implanta antes de una transección de la médula espinal. Una limitación de la Telemet descritoRY procedimiento es que la implantación en la arteria femoral puede influir en el suministro de sangre a la extremidad posterior ipsilateral.

Utilizando la técnica de telemetría antes descrito, se registró con éxito los parámetros cardiovasculares en la médula espinal lesionada animales. En los animales con solo SCI, MAP se redujo significativamente, mientras que HR aumentó en comparación con los animales no tratados previamente, en consonancia con los informes anteriores 9. En los animales con BS-NSC injerto, PAM y FC acercado niveles medidos en los animales no tratados previamente (Figura 2). Durante la distensión colorr...

Watch this Scientific Journal Video about A Radio-telemetric System to Monitor Cardiovascular Function in Rats with Spinal Cord Transection and Embryonic Neural Stem Cell Grafts at JoVE.com

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

Se presenta un protocolo para el uso de un sistema de radio-telemétrico para registrar los parámetros cardiovasculares en la T4 de la médula espinal de ratas cortados transversalmente ocho semanas después del tronco cerebral embrionaria de células madre neurales de injerto en el sitio de la lesión. La telemetría es una técnica avanzada para evaluar con precisión la función cardiovascular en conscientes de la médula espinal de ratas lesionadas con libertad de movimientos.

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 ...

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10.4K vistas.  Drexel University College of Medicine.  Se presenta un protocolo para el uso de un sistema de radio-telemétrico para registrar los parámetros cardiovasculares en la T4 de la médula espinal de ratas cortados transversalmente ocho semanas después del tronco cerebral embrionaria de células madre neurales de injerto en el sitio de la lesión. La telemetría es una técnica avanzada para evaluar con precisión la función cardiovascular en conscientes de la médula espinal de ratas lesionadas con libertad de m...

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

Assessing Endothelial Vasodilator Function with the Endo-PAT 2000

The endothelium is a delicate monolayer of cells that lines all blood vessels, and which comprises the systemic and lymphatic capillaries. By virtue of the panoply of paracrine factors that it secretes, the endothelium regulates the contractile and proliferative state of the underlying vascular smooth muscle, as well as the interaction of the vessel wall with circulating blood elements. Because of its central role in mediating vessel tone and growth, its position as gateway to circulating immune cells, and its local regulation of hemostasis and coagulation, the the properly functioning endothelium is the key to cardiovascular health. Conversely, the earliest disorder in most vascular diseases is endothelial dysfunction.
In the arterial circulation, the healthy endothelium generally exerts a vasodilator influence on the vascular smooth muscle. There are a number of methods to assess endothelial vasodilator function. The Endo-PAT 2000 is a new device that is used to assess endothelial vasodilator function in a rapid and non-invasive fashion. Unlike the commonly used technique of duplex ultra-sonography to assess flow-mediated vasodilation, it is totally non-operator-dependent, and the equipment is an order of magnitude less expensive. The device records endothelium-mediated changes in the digital pulse waveform known as the PAT ( peripheral Arterial Tone) signal, measured with a pair of novel modified plethysmographic probes situated on the finger index of each hand. Endothelium-mediated changes in the PAT signal are elicited by creating a downstream hyperemic response. Hyperemia is induced by occluding blood flow through the brachial artery for 5 minutes using an inflatable cuff on one hand. The response to reactive hyperemia is calculated automatically by the system. A PAT ratio is created using the post and pre occlusion values. These values are normalized to measurements from the contra-lateral arm, which serves as control for non-endothelial dependent systemic effects. Most notably, this normalization controls for fluctuations in sympathetic nerve outflow that may induce changes in peripheral arterial tone that are superimposed on the hyperemic response.
In this video we demonstrate how to use the Endo-PAT 2000 to perform a clinically relevant assessment of endothelial vasodilator function.

A noninvasive procedure to assess endothelial function is demonstrated using the Endo-PAT 2000.

Valoración de la función endotelial vasodilatador con el 2000 Endo-PAT

The endothelium is a delicate monolayer of cells that lines all blood vessels, and which comprises the systemic and lymphatic capillaries. By virtue of ...

Investigación

Medicina

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 ...

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 ...

Synergetic Use of Neural Precursor Cells and Self-assembling Peptides in Experimental Cervical Spinal Cord Injury

Spinal cord injuries (SCI) cause serious neurological impairment and psychological, economic, and social consequences for patients and their families. Clinically, more than 50% of SCI affect the cervical spine1. As a consequence of the primary injury, a cascade of secondary mechanisms including inflammation, apoptosis, and demyelination occur finally leading to tissue scarring and development of intramedullary cavities2,3. Both represent physical and chemical barriers to cell transplantation, integration, and regeneration. Therefore, shaping the inhibitory environment and bridging cavities to create a supportive milieu for cell transplantation and regeneration is a promising therapeutic target4. Here, a contusion/compression model of cervical SCI using an aneurysm clip is described. This model is more clinically relevant than other experimental models, since complete transection or ruptures of the cord are rare. Also in comparison to the weight drop model, which in particular damage the dorsum columns, circumferential compression of the spinal cord appears advantageous. Clip closing force and duration can be adjusted to achieve different injury severity. A ring spring facilitates precise calibration and constancy of clip force. Under physiological conditions, synthetic self-assembling peptides (SAP) self-assemble into nanofibers and thus, are appealing for application in SCI5. They can be injected directly into the lesion minimizing damage to the cord. SAPs are biocompatible structures erecting scaffolds to bridge intramedullary cavities and thus, equip the damaged cord for regenerative treatments. K2(QL)6K2 (QL6) is a novel SAP introduced by Dong et al.6 In comparison to other peptides, QL6 self-assembles into &#946;-sheets at neutral pH6.14 days after SCI, after the acute stage, SAPs are injected into the center of the lesion and neural precursor cells (NPC) are injected into adjacent dorsal columns. In order to support cell survival, transplantation is combined with continuous subdural administration of growth factors by osmotic micro pumps for 7 days.

Treating cervical spinal cord injury with both self-assembling peptides (SAP) and neural precursor cells (NPC), together with growth factors, is a promising approach to promote regeneration and recovery. A contusion/compression aneurysm clip rat model of cervical SCI and combined treatment involving SAP injection and NPC transplantation is established.

El uso sinérgico de las células precursoras neuronales y Péptidos de autoensamblaje en Experimental cervical Lesión de la Médula Espinal

Spinal cord injuries (SCI) cause serious neurological impairment and psychological, economic, and social consequences for patients and their families. ...

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 ...

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.

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) ...

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 ...

Vein Interposition Model: A Suitable Model to Study Bypass Graft Patency

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. Research models for bypass graft failure are essential for a better understanding of pathobiological and pathophysiological processes during graft patency loss. Large animal models, such as pigs or sheep, resemble human anatomical structures but require special facilities and equipment. This video describes a rat vein interposition model to investigate vein graft patency loss. Rats are inexpensive and easy to handle. Compared to mouse models, the convenient size of rats permits better operability and enables a sufficient amount of material to be obtained for further diverse analysis. In brief, the inferior epigastric vein of a donor rat is harvested and used to replace a segment of the femoral artery. Anastomosis is conducted via single stitches and sealed with fibrin glue. Graft patency can be monitored non-invasively using duplex sonography. Myointimal hyperplasia, which is the main cause for graft patency loss, develops progressively over time and can be calculated from histological cross sections.

This video demonstrates a model to study the development of myointimal hyperplasia after venous interposition surgery in rats.

La interposición de la vena del modelo: un modelo adecuado para estudiar la permeabilidad del injerto de bypass

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. ...

Studying the Hypothalamic Insulin Signal to Peripheral Glucose Intolerance with a Continuous Drug Infusion System into the Mouse Brain

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues contributes to type 2 diabetes mellitus (T2DM) development and appetite regulation in the hypothalamus. Chemokine CCL5 and C-C chemokine receptor type 5 (CCR5) levels have been suggested to mediate arteriosclerosis and glucose intolerance in type 2 diabetes mellitus (T2DM). In addition, CCL5 plays a neuroendocrine role in the hypothalamus by regulating food intake and body temperature, thus, prompting us to investigate its function in hypothalamic insulin signaling and the regulation of peripheral glucose metabolism.
The micro-osmotic pump brain infusion system is a quick and precise way to manipulate CCL5 function and study its effect in the brain. It also provides a convenient alternative approach to generating a transgenic knockout animal. In this system, CCL5 signaling was blocked by intracerebroventricular (ICV) infusion of its antagonist, MetCCL5, using a micro-osmotic pump. The peripheral glucose metabolism and insulin responsiveness was detected by the Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT). Insulin signaling activity was then analyzed by protein blot from tissue samples derived from the animals.
After 7-14 days of MetCCL5 infusion, the glucose metabolism and insulin responsiveness was impaired in mice, as seen in the results of the OGTT and ITT. The IRS-1 serine302 phosphorylation was increased and the Akt activity was reduced in mice hypothalamic neurons following CCL5 inhibition. Altogether, our data suggest that blocking CCL5 in the mouse brain increases the phosphorylation of IRS-1 S302 and interrupts hypothalamic insulin signaling, leading to a decrease in insulin function in peripheral tissues as well as the impairment of glucose metabolism.

This protocol studies the role of chemokine (C-C motif) ligand 5 (CCL5) in the hypothalamus by delivering an antagonist, MetCCL5, into the mouse brain using a micro-osmotic pump brain infusion system. This transient inhibition of CCL5 activity interrupted hypothalamic insulin signaling, leading to glucose intolerance and peripheral systemic insulin sensitivity.

Estudio de la señal de insulina hipotalámica a la intolerancia de la glucosa periférica con un sistema de infusión continua de medicamentos en el cerebro de ratón

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues ...

Balloon-based Injury to Induce Myointimal Hyperplasia in the Mouse Abdominal Aorta

The use of animal models is essential for a better understanding of MH, one major cause for arterial stenosis.In this article, we demonstrate a murine balloon denudation model, which is comparable with established vessel injury models in large animals. The aorta denudation model with balloon catheters mimics the clinical setting and leads to comparable pathobiological and physiological changes. Briefly, after performing a horizontal incision in the aorta abdominalis, a balloon catheter will be inserted into the vessel, inflated, and introduced retrogradely. Inflation of the balloon will lead to intima injury and overdistension of the vessel. After removing the catheter, the aortic incision will be closed with single stiches. The model shown in this article is reproducible, easy to perform, and can be established quickly and reliably. It is especially suitable for evaluating expensive experimental therapeutic agents, which can be applied in an economical fashion. By using different knockout-mouse strains, the impact of different genes on MH development can be assessed.

This article demonstrates a murine model to study the development of myointimal hyperplasia (MH) after aortic balloon injury.

Lesión basada en globo para inducir la hiperplasia Myointimal de la Aorta Abdominal de ratón

The use of animal models is essential for a better understanding of MH, one major cause for arterial stenosis.In this article, we demonstrate a murine ...