Nos gustaría agradecer el apoyo financiero para este trabajo de los Institutos Canadienses de Investigación en Salud (CIHR), la Fundación Krembil Familia, el Presidente Halbert en Neural Repair and Regeneration, Phillip y Peggy DeZwirek, y Gordon Yao por la contribución a la figura 2 . Klaus Zweckberger fue financiado por una beca de la &quot;Deutsche Forschungsgesellschaft&quot; (DFG).

NOTA: El siguiente protocolo experimental fue aprobado por el comité de cuidado de los animales de la Red de Salud de la Universidad (Toronto, Canadá) y está de acuerdo con las políticas establecidas en la guía para el cuidado y uso de animales de experimentación preparados por el Consejo Canadiense de cuidado de los animales . 1. Cervical Contusión Aneurisma Clip / Compresión Modelo <ol><li> Antes de instrumentos de autoclave de cirugía y mantener condiciones estériles durante todo el procedimiento surgcial poniendo los instrumentos en un baño de alcohol al 70%. </li><li> Anestesiar ratas Wistar (250-270 g) con una combinación de oxígeno (O 2), óxido nitroso (N 2 O) (1: 1), y 1.8 a 2.2% isoflurano y apoyar la respiración espontánea a través de una máscara de anestesia de gas. Para la inducción de la anestesia comenzar con 5% de isoflurano durante 1 minuto y reducir después. Antes de comenzar la cirugía, la profundidad de control de la anestesia dando un estímulo doloroso (por ejemplo. En las patas). Aplicar pomadas grasasen los ojos para evitar la sequedad y para prevenir las infecciones posteriores. </li><li> Poner las ratas en un cojín de calentamiento (37 ° C) y fijar la cabeza en un marco estereotáctico. </li><li> Afeitarse la región quirúrgica alrededor de la columna vertebral cervical y desinfectar con yodo povidona y 70% de alcohol. </li><li> Hacer una incisión en la línea media por encima de la columna vertebral de la vértebra cervical (C2) que llega hasta el processus prominente del cuerpo vertebral torácica 2 (T2). </li><li> Corte a través de la capa externa de los músculos vertebrales directamente en la línea media (para evitar el sangrado) en una dirección cráneo-caudal y más diseccionar las capas musculares más profundas sin rodeos hasta llegar a la spinosus processus y las láminas. Inserte retractores. </li><li> Orientar la spinosus processus prominente de la T2 cuerpo vértebra para observar los niveles previstos para laminectomía. Después de la identificación y preparación micro-quirúrgica de las láminas seleccionado, cortar a través de la flavae ligamenti para aflojar las láminas y la spinosus processus. Por último, cut a través de las láminas con un lateral de las podadoras de hueso a la médula espinal y eliminar aquellos suavemente, evitando cualquier compresión de la médula espinal en sí. NOTA: Los más comunes son los niveles C5 / 6, C6 / 7, o C7 / T1. La tasa de mortalidad perioperatoria aumenta cuanto más rostral del nivel de la lesión. Sangrado del seno venoso paravertebral es común y puede abordarse mediante la compresión cuidado con esponja. </li><li> Antes de insertar el clip para traumatizar el cable, identificar nuevas raíces nerviosas para librarlos de recorte (especialmente a nivel C5 / 6). </li><li> Con el fin de garantizar el buen inducción clip, aflojar la duramadre ventral de la cara dorsal de los cuerpos vertebrales con un gancho y preparar un corredor para el clip. </li><li> Por último, inserte el clip abierto y dejar que se cierran de golpe (cierre rápido) para lograr una lesión por contusión. Fuerza de cierre del clip y la duración de cierre de clip determinan la intensidad del trauma y el grado de compresión. Comúnmente utilizados son fuerzas de clip de entre 15 a 35 g y un clipping duración de, por ejemplo 1 min. (Figura 1). </li><li> Después de la retirada de la pinza, adaptar músculos de 2 capas y cerrar la herida. </li><li> Deje de anestesia y deja la estela de los animales bajo su observación continua hasta que se recupera la conciencia suficiente para decúbito esternal. Por último, poner la rata en una jaula individual y seguir las pautas de tratamiento post-operatorio. </li><li> Puesto que los animales luchan con la gravedad de este tipo de lesión, debe prestar especial atención a los tratamientos post-operatorios: <ol><li> Administrar analgésicos (buprenorfina y meloxicam durante 3 días y 5 días, respectivamente y según los síntomas clínicos). </li><li> Dé subcutánea solución salina adicional para 3 días (2 veces al día, 5-10 mililitro (ml)). </li><li> Proporcionar antibióticos en el agua de bebida 2 días antes y hasta 7 días después de la cirugía (por ejemplo Moxifloxacina) </li><li> Apriete la vejiga urinaria 2-3 veces al día hasta que la recuperación de la función de la vejiga es constanteLy aparente. </li><li> Observar los déficits neurológicos y estado fisiológico de los animales operados por lo menos una vez al día. </li></ol></li></ol> 2. La inyección de los PAE y NPCs (14 días después de la lesión) <ol><li> Inducir la anestesia como se describe en 1.1. a 1,3, fijar la cabeza de la rata en un marco estereotáxico, retire los puntos de sutura o grapas de la herida, y desinfectar la herida y el área quirúrgica con povidona yodada y el 70% de alcohol. </li><li> Diseccionar cuidadosamente los músculos paravertebrales, inserte retractores, eliminar el tejido cicatrizal microscópicamente de la duramadre, y volver a exponer el sitio de la lesión. </li><li> Preparar los PAE en una concentración de 1% (w / v) para realizar un gel de matriz extracelular. PAE QL6 tienen un pH fisiológicamente compatibles y no necesitan ser tamponada antes de la inyección. Para la visualización de los PAE en la médula espinal, utilizar un derivado fluorescente de QL6 (QL6-FITC). </li><li> Inyectar PAE (5 microlitros (l) en el centro de la lesión, distribuidos en 2 porciones, cada uno2,5 l bilateral de la línea media. Utilice una jeringa Hamilton conectado al marco estereotáctico con un micro capilar de vidrio (100 micrómetros (micras) de diámetro exterior (OD)). Abrir la duramadre cuidadosamente con la punta de una aguja afilada, e insertar el capilar de vidrio estereotácticamente 2 milímetros (mm) en la médula espinal traumatizada. </li><li> Después de la inyección de un tercio del volumen, retire la aguja de 1,5 mm de profundidad, y después de otros 1/3 a 1 mm. Después de la inyección de todo el volumen, y antes de la retirada de la jeringa, esperar 5 minutos para estabilizar la formación de gel. </li><li> Para generar NPCs, utilizar ratones adultos DsRed (o YFP ratones positivos, verde) y aislar y cultivar ellos desde la zona paraventrical 4,18,21. </li><li> Evaluar la viabilidad de NPC por tinción de azul de tripano que indican la presencia de ~ 90% de células vivas en la suspensión celular. Diluir las células en medio de crecimiento (50 x 103 células vivas / l) y luego usarlos para el trasplante de células. </li><li> Haga cuatro 2 l (8 l vo total delume, que contiene 4 x 10 5 NPCs) inyecciones intraespinales bilateralmente a los 2 mm rostral y caudal de la zona de la lesión. Después de la apertura de la duramadre, inserte el micro capilar de vidrio Hamilton 1,5 mm por debajo de la superficie dorsal de la médula espinal e inyectar 2 l de la suspensión celular. Elija una velocidad de inyección a aproximadamente 0,5 l / min (min). </li><li> Al final de cada inyección y antes de la retirada del capilar de la cuerda, espere por lo menos 1 min permitiendo que el tejido se extiende para acomodar el nuevo volumen de la celda. (Figura 2) </li></ol> 3. Implantación de subdural Bombas para el Factor de Crecimiento de aplicaciones <ol><li> Con el fin de enriquecer el fluido espinal cerebral (CSF) con factores de crecimiento apoyar la supervivencia celular, usar bombas de micro-dilución osmótica factores de crecimiento sub-durally largo de 7-14 días, con una tasa de dilución de 0,5 l / h, un diámetro de catéter de 0,04 cm OD, y un volumen de embalse de 100 l. </li><li> Elija factores de crecimiento preferidas(Por ejemplo, el factor de crecimiento derivado del cerebro (BDGF), factor de crecimiento epidérmico (EGF), factor de crecimiento de fibroblastos (FGF)), llenar las bombas 6-10 hr implantación previa, y equilibre las bombas en un baño de agua a 37 ° C. </li><li> Inmediatamente después de las inyecciones de la APN, preparar una cavidad subcutánea para colocar la bomba. Lugares preferidos son los flancos laterales de toracolumbar región abdominal evitando importante malestar local causado por la propia bomba. </li><li> Para obtener la mayor concentración de factores de crecimiento, asegúrese de que la punta abierta del catéter termina cerca del lugar de la lesión. Por lo tanto, realice un salto-laminectomía del nivel superior o inferior adyacente. Por ejemplo, si el SCI es en C7 / T1, realizar una pequeña laminectomía de C5. </li><li> Ponga la bomba en el hueco subcutáneo, acortar el catéter a la longitud necesaria, y seguro que con varias suturas (6.0) en los músculos paravertebrales, evitando cualquier movimiento luxación asociada. </li><li> Después de abrir la duramadre (por ejemplo, en C5) con la punta afiladade una aguja, introducir el catéter en el espacio subdural y dejar que se deslice en una dirección caudal sin ninguna resistencia y sin dañar el cable. La lámina omitido de, por ejemplo C6 sirve punto de fijación y estabilización del catéter adicional. </li><li> Asegúrese de que el catéter se ejecuta sin problemas y no se pliega. </li><li> Cerrar músculos por capa, y la piel, con suturas o grapas. (Figura 3) </li><li> Deje de anestesia y deja la estela de los animales bajo su observación continua hasta que se recupera la conciencia suficiente para decúbito esternal. Por último, volver a ponerlos en una sola jaula y seguir las pautas de tratamiento post-operatorio. </li><li> Proporcionar tratamiento postoperatorio especial, a pesar de que, los animales podrían haber recuperado en parte en este punto del tiempo: <ol><li> Administrar analgésicos (Buprenorthine y meloxicam durante 3 días y 5 días, respectivamente y según los síntomas clínicos). </li><li> Proporcionar antibióticos en las botellas de agua 2 días antes hasta7 días después de la cirugía (por ejemplo Moxifloxacina). </li><li> Siga apretando la vejiga urinaria, si sigue siendo necesaria. </li><li> Déle líquidos subcutáneos adicionales, si las ratas parecen deshidratado. </li><li> Mantenga la observación de la función neurológica y la condición fisiológica de los animales operados al menos una vez al día. </li><li> Administrar tratamiento inmunosupresor 2 días antes de la inyección de la APN y hasta 7 días después transplanation (minociclina) y hasta el sacrificio (Sandimmune), respectivamente. </li></ol></li></ol> Evaluación 4. Tejido <ol><li> Sacrificar los animales en el final del tiempo de observación (por ejemplo. 4 semanas después de SCI) en anestesia profunda (5% de isoflurano durante 2-3 min) y perfundir ellos transcardially con 50 ml de frío (4 ° C) solución salina seguido por 150 ml de frío 4 % de paraformaldehído en 0,1 M de solución salina tamponada con fosfato (PBS). </li><li> Retire la médula espinal y lo puso en el 4% de paraformaldehído en 0,1 M de buffer fosfato salino (PBS) durante 24 horas. </li><li&gt; Poner la médula espinal en solución de sacarosa al 25% para crioprotección durante 48 horas. </li><li> Proporcionar cryosections longitudinales con un espesor &lt;30 micras. </li><li> Para la tinción inmunohistoquímica de todos los celulares de los núcleos que proporcionan un uso del fondo DAPI (1: 1000). NPCs DsRed positivos aparecen rojos, QL-6 FITC aparece en verde, y ambos lo tanto no necesita ser manchado especialmente. (Figura 4). </li><li> Por microscopía electrónica de barrido (SEM) permiten muestras remojo en glutaraldehído a 4 ° C durante 2 horas, se deshidratan lentamente en pasos de 10% de incremento de etanol durante 5 minutos y los colocan en un líquido presurizado CO 2 sifón durante 1 hora. Andamios capa con oro utilizando un dispositivo de recubrimiento por pulverización catódica. Tome imágenes con un microscopio electrónico de barrido Hitachi S-3400N. (Figura 5) </li></ol>

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Covidien supplied the QL6 SAP for this study.

Este protocolo ha sido desarrollado para permitir al lector a realizar un modelo de lesión cervical en ratas y de utilizar un enfoque de tratamiento combinado con programas de ajuste estructural y NPCs que promueven una mejor recuperación después de la lesión cervical. En particular, en comparación con otros modelos de trauma cervical, como la (hemi) -transection modelo o los modelos de caída de peso y de concusión, el modelo de clip contusión / compresión representa tanto principal...

Más del 50% de las lesiones de la médula espinal están relacionadas con la columna cervical. En el contexto clínico dos principales mecanismos fisiopatológicos se describen: la contusión inicial de la médula espinal y posteriormente, la compresión en curso causada por fracturas de huesos, hemorragias o inflamación del tejido. El clip de aneurisma imita contusión modelo / compresión de ambos mecanismos fisiopatológicos: rompiendo el clip produce una contusión y la duración de recorte representa el componente de compresión, admitiendo que la compresión en entornos clínicos causados ​​por fracturas de huesos, hemorragias o hinchazón de los tejidos última significativa más tiempo. El clip de aneurisma utilizado es modificado por un resorte anillo garantizando la fuerza exacta y reproducible de recorte. Sobre todo en comparación con el hemi-transección o el modelo de contusión, este clip de aneurisma imita modelo mejores entornos clínicos. Mientras que los pacientes con lesiones torácicas sufren de paraplejía, la mayoría de los pacientes con inj cervicaluries son tetrapléjico y completamente dependiente. La estructura anatómica de la médula cervical, sin embargo, muestra diferencias significativas en comparación con el dorsal o lumbar, y por lo tanto, se trata en particular, en este protocolo. El desarrollo de cavidades intramedulares y cicatrices en los tejidos son obstáculos para la recuperación y regeneración. Para superar estas barreras el uso de material de soporte es un enfoque prometedor. Péptidos de autoensamblaje pueden inyectarse directamente en el epicentro de la lesión. Allí se ensamblan para formar andamios nano-fibra puente de la cavidad y mejorar el medio ambiente inhibitorio al reducir la inflamación y cicatrización de los tejidos. Mientras que los materiales rígidos causan un daño considerable de la médula espinal durante la implantación, los péptidos de fluidos pueden ser inyectados de forma segura y sin daño adicional grave. La mejora del entorno inhibitorio con péptidos auto-montaje antes del trasplante de células madre, por lo tanto, apoyar la celda iNTEGRACIÓN, la diferenciación y, finalmente, la recuperación funcional, después de la lesión de la médula espinal cervical.

<table border="0" cellpadding="0" cellspacing="0" width="541">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Name</td>
			<td style="width:268px;">Company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aneurysmal clip</td>
			<td>SharpTech</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical microscope</td>
			<td>Leica</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro injection system</td>
			<td>World Precision Instruments, Inc.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Small animal stereotaxic instrument</td>
			<td>David Kopf Instruments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hamilton syringe</td>
			<td>Hamilton company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Subdural pumps</td>
			<td>Alzet osmotic micro pump 1007D</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical instrument</td>
			<td>Fine Science tools</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoflurane USP</td>
			<td>Pharmaceutical Partners of Canada Inc.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.9% Sodium Chloride injection USP</td>
			<td>Baxter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">7.5% Povidone iodine</td>
			<td>Purdue Pharma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70% Isopropyl alcohol USP</td>
			<td>GreenField Ethanol Inc.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QL6 SAP</td>
			<td>Covidien</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.4% Trypan blue</td>
			<td>Gibco</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Platelet-Derived Growth Factor (PDGF)</td>
			<td>Sigma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Epidermal Growth Factor (EGF)</td>
			<td>Sigma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fibroblast Growth Factor (FGF)</td>
			<td>Sigma</td>
		</tr>
	</tbody>
</table>

synergetic use of neural precursor cells and self-assembling peptides in experimental cervical spinal cord injury

Lesiones de la médula espinal (SCI) causan deterioro neurológico grave y las consecuencias psicológicas, económicas y sociales para los pacientes y sus familias. Clínicamente, más de 50% de SCI afecta a la columna cervical 1. Como consecuencia de la lesión primaria, una cascada de mecanismos secundarios incluyendo la inflamación, apoptosis, y la desmielinización se producen finalmente conduce a la cicatrización de tejidos y el desarrollo de cavidades intramedulares 2,3. Ambos representan barreras físicas y químicas en el trasplante de células, la integración, y la regeneración. Por lo tanto, la configuración del entorno inhibitorio y la reducción de cavidades para crear un medio de apoyo para el trasplante de células y la regeneración es un objetivo terapéutico prometedor 4. Aquí, se describe un modelo de contusión / compresión del SCI cervical usando un clip de aneurisma. Este modelo es más clínicamente relevante que otros modelos experimentales, ya que la transección completa o rupturas del cable son raros. También en COMPARACIÓNn para el modelo de caída de peso, que, en particular, el daño de las columnas dorso, la compresión circunferencial de la médula espinal parece ventajoso. Fuerza de cierre de clip y la duración se puede ajustar para lograr gravedad de la lesión diferente. Un muelle de anillo facilita la calibración precisa y la constancia de la fuerza clip. En condiciones fisiológicas, los péptidos de autoensamblaje sintéticos (SAP) auto-ensamblarse en nanofibras y, por tanto, están haciendo un llamado para la aplicación en SCI 5. Ellos pueden ser inyectados directamente en la lesión minimizar daños en el cable. PAE son estructuras biocompatibles erigir andamios para colmar cavidades intramedulares y, por tanto, equipar el cable dañado por tratamientos regenerativos. K2 (QL) 6K2 (QL6) es una novela SAP introducido por Dong et a l. 6 En comparación con otros péptidos, QL6 auto-ensambla en β-hojas a pH neutro 6 0,14 días después de la lesión, después de la fase aguda, los PAE se inyectan en el centro de la lesión y las células precursoras neurales (NPC) son injected en columnas dorsales adyacentes. Con el fin de apoyar la supervivencia celular, el trasplante se combina con la administración subdural continua de factores de crecimiento por medio de bombas micro osmóticos para 7 días.

<html> Al realizar el procedimiento anteriormente descrito, obtendrá un andamio SAP cerrar la cavidad y que ofrece una mejora del medio ambiente inhibitorio, menos cicatrización del tejido y un aumento en la supervivencia NPC. Figura 4 muestra una sección longitudinal de una médula espinal de rata obtenido en el sitio de la lesión 6 semanas después de SCI y 4 semanas después de la inyección QL6 SAP y NPC trasplante. Péptidos QL6 fueron inyectados con éxito en el cable, agregada en el epicentro ...

Watch this Scientific Journal Video about Synergetic Use of Neural Precursor Cells and Self-assembling Peptides in Experimental Cervical Spinal Cord Injury at JoVE.com

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

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. ...

synergetic-use-neural-precursor-cells-self-assembling-peptides

Research

JoVE Journal

Medicine

9.1K vistas.  University Health Network, Krembil Neuroscience Centre. 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. 

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

Intraspinal Cell Transplantation for Targeting Cervical Ventral Horn in Amyotrophic Lateral Sclerosis and Traumatic Spinal Cord Injury

Respiratory compromise due to phrenic motor neuron loss is a debilitating consequence of a large proportion of human traumatic spinal cord injury (SCI) cases 1 and is the ultimate cause of death in patients with the motor neuron disorder, amyotrophic laterals sclerosis (ALS) 2.
ALS is a devastating neurological disorder that is characterized by relatively rapid degeneration of upper and lower motor neurons. Patients ultimately succumb to the disease on average 2-5 years following diagnosis because of respiratory paralysis due to loss of phrenic motor neuron innnervation of the diaphragm 3. The vast majority of cases are sporadic, while 10% are of the familial form. Approximately twenty percent of familial cases are linked to various point mutations in the Cu/Zn superoxide dismutase 1 (SOD1) gene on chromosome 21 4. Transgenic mice 4,5 and rats 6 carrying mutant human SOD1 genes (G93A, G37R, G86R, G85R) have been generated, and, despite the existence of other animal models of motor neuron loss, are currently the most highly used models of the disease.
Spinal cord injury (SCI) is a heterogeneous set of conditions resulting from physical trauma to the spinal cord, with functional outcome varying according to the type, location and severity of the injury 7. Nevertheless, approximately half of human SCI cases affect cervical regions, resulting in debilitating respiratory dysfunction due to phrenic motor neuron loss and injury to descending bulbospinal respiratory axons 1. A number of animal models of SCI have been developed, with the most commonly used and clinically-relevant being the contusion 8.
Transplantation of various classes of neural precursor cells (NPCs) is a promising therapeutic strategy for treatment of traumatic CNS injuries and neurodegeneration, including ALS and SCI, because of the ability to replace lost or dysfunctional CNS cell types, provide neuroprotection, and deliver gene factors of interest 9.
Animal models of both ALS and SCI can model many clinically-relevant aspects of these diseases, including phrenic motor neuron loss and consequent respiratory compromise 10,11. In order to evaluate the efficacy of NPC-based strategies on respiratory function in these animal models of ALS and SCI, cellular interventions must be specifically directed to regions containing therapeutically relevant targets such as phrenic motor neurons. We provide a detailed protocol for multi-segmental, intraspinal transplantation of NPCs into the cervical spinal cord ventral gray matter of neurodegenerative models such as SOD1G93A mice and rats, as well as spinal cord injured rats and mice 11.

Neural precursor transplantation is a promising strategy for protecting and/or replacing lost/dysfunctional cervical phrenic motor neurons in spinal cord injury (SCI) and the motor neuron disorder, amyotrophic laterals sclerosis (ALS). We provide a protocol for cell delivery to cervical spinal cord ventral horn in rodent models of ALS and SCI.

Intraespinal trasplante de células de Orientación asta ventral del cuello uterino en la esclerosis lateral amiotrófica y la lesión de la médula espinal

Respiratory compromise due to phrenic motor neuron loss is a debilitating consequence of a large proportion of human traumatic spinal cord injury (SCI) ...

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 Contusive Model of Unilateral Cervical Spinal Cord Injury Using the Infinite Horizon Impactor

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of spinal cord injury (SCI) in which the thoracic spinal cord is injured. Additionally, because most human cord injuries occur as the result of blunt, non-penetrating trauma (e.g. motor vehicle accident, sporting injury) where the spinal cord is violently struck by displaced bone or soft tissues, the majority of SCI researchers are of the opinion that the most clinically relevant injury models are those in which the spinal cord is rapidly contused.1 Therefore, an important step in the preclinical evaluation of novel treatments on their way to human translation is an assessment of their efficacy in a model of contusion SCI within the cervical spinal cord. Here, we describe the technical aspects and resultant anatomical and behavioral outcomes of an unilateral contusive model of cervical SCI that employs the Infinite Horizon spinal cord injury impactor.
Sprague Dawley rats underwent a left-sided unilateral laminectomy at C5. To optimize the reproducibility of the biomechanical, functional, and histological outcomes of the injury model, we contused the spinal cords using an impact force of 150 kdyn, an impact trajectory of 22.5&deg; (animals rotated at 22.5&deg;), and an impact location off of midline of 1.4 mm. Functional recovery was assessed using the cylinder rearing test, horizontal ladder test, grooming test and modified Montoya's staircase test for up to 6 weeks, after which the spinal cords were evaluated histologically for white and grey matter sparing.
The injury model presented here imparts consistent and reproducible biomechanical forces to the spinal cord, an important feature of any experimental SCI model. This results in discrete histological damage to the lateral half of the spinal cord which is largely contained to the ipsilateral side of injury. The injury is well tolerated by the animals, but does result in functional deficits of the forelimb that are significant and sustained in the weeks following injury. The cervical unilateral injury model presented here may be a resource to researchers who wish to evaluate potentially promising therapies prior to human translation.

A reliable and repeatable way to produce a cervical unilateral spinal cord injury using the Infinite Horizon impactor is described. The method takes advantage of a custom designed frame and clamp to stabilize the spine. The standardized procedure and biomechanical injury parameters result in sufficient and sustained injuries.

Un modelo de la contundente unilateral Lesión Medular Cervical Uso del Impactador Infinite Horizon

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of ...

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 ...

Evaluation of Respiratory Muscle Activation Using Respiratory Motor Control Assessment (RMCA) in Individuals with Chronic Spinal Cord Injury

During breathing, activation of respiratory muscles is coordinated by integrated input from the brain, brainstem, and spinal cord. When this coordination is disrupted by spinal cord injury (SCI), control of respiratory muscles innervated below the injury level is compromised1,2 leading to respiratory muscle dysfunction and pulmonary complications. These conditions are among the leading causes of death in patients with SCI3. Standard pulmonary function tests that assess respiratory motor function include spirometrical and maximum airway pressure outcomes: Forced Vital Capacity (FVC), Forced Expiratory Volume in one second (FEV1), Maximal Inspiratory Pressure (PImax) and Maximal Expiratory Pressure (PEmax)4,5. These values provide indirect measurements of respiratory muscle performance6. In clinical practice and research, a surface electromyography (sEMG) recorded from respiratory muscles can be used to assess respiratory motor function and help to diagnose neuromuscular pathology. However, variability in the sEMG amplitude inhibits efforts to develop objective and direct measures of respiratory motor function6. Based on a multi-muscle sEMG approach to characterize motor control of limb muscles7, known as the voluntary response index (VRI)8, we developed an analytical tool to characterize respiratory motor control directly from sEMG data recorded from multiple respiratory muscles during the voluntary respiratory tasks. We have termed this the Respiratory Motor Control Assessment (RMCA)9. This vector analysis method quantifies the amount and distribution of activity across muscles and presents it in the form of an index that relates the degree to which sEMG output within a test-subject resembles that from a group of healthy (non-injured) controls. The resulting index value has been shown to have high face validity, sensitivity and specificity9-11. We showed previously9 that the RMCA outcomes significantly correlate with levels of SCI and pulmonary function measures. We are presenting here the method to quantitatively compare post-spinal cord injury respiratory multi-muscle activation patterns to those of healthy individuals.

Evaluation of Respiratory Muscle Activation Using Respiratory Motor Control Assessment RMCA in Individuals with Chronic Spinal Cord Injury

The purpose of this publication is to present our original work on a multi-muscle surface electromyographic approach to quantitatively characterize respiratory muscle activation patterns in individuals with chronic spinal cord injury using vector-based analysis.

Evaluación de activación muscular respiratoria Utilizar evaluaciones de control de motores respiratoria (RMCA) en personas con lesión de la médula espinal crónica

During breathing, activation of respiratory muscles is coordinated by integrated input from the brain, brainstem, and spinal cord. When this coordination ...

A Murine Model of Cervical Spinal Cord Injury to Study Post-lesional Respiratory Neuroplasticity

A cervical spinal cord injury induces permanent paralysis, and often leads to respiratory distress. To date, no efficient therapeutics have been developed to improve/ameliorate the respiratory failure following high cervical spinal cord injury (SCI). Here we propose a murine pre-clinical model of high SCI at the cervical 2 (C2) metameric level to study diverse post-lesional respiratory neuroplasticity. The technique consists of a surgical partial injury at the C2 level, which will induce a hemiparalysis of the diaphragm due to a deafferentation of the phrenic motoneurons from the respiratory centers located in the brainstem. The contralateral side of the injury remains intact and allows the animal recovery. Unlike other SCIs which affect the locomotor function (at the thoracic and lumbar level), the respiratory function does not require animal motivation and the quantification of the deficit/recovery can be easily performed (diaphragm and phrenic nerve recordings, whole body ventilation). This pre-clinical C2 SCI model is a powerful, useful, and reliable pre-clinical model to study various respiratory and non-respiratory neuroplasticity events at different levels (molecular to physiology) and to test diverse putative therapeutic strategies which might improve the respiration in SCI patients.

Respiratory failure is the leading cause of death following a cervical spinal cord injury. Having a reproducible, quantifiable, and reliable pre-clinical animal model of respiratory failure induced by a partial cervical injury will help to understand the subsequent respiratory and non-respiratory neuroplasticity and allow testing putative repair strategies.

Un modelo murino de la médula espinal cervical Lesión en estudio post-lesional Respiratorio Neuroplasticidad

A cervical spinal cord injury induces permanent paralysis, and often leads to respiratory distress. To date, no efficient therapeutics have been developed ...

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 ...