El desarrollo de este dispositivo fue apoyada por el LISA Co., Louisville, Kentucky. También reconocemos el apoyo continuo de Norton Healthcare, Louisville, KY a CBS y NIH NS050243, NS052290 y NS059622 a XMX.

1. Preparación de los animales y la aplicación del estabilizador de la columna vertebral El ratón de la columna cervical es cóncava ventralmente como se ve desde la vista lateral. Las apófisis espinosas de C3 a T1 son pequeñas y friable y, por lo tanto, no son adecuados para la estabilización vertebral comúnmente descrito como 3,4. Recomendamos estabilización de la columna se realizó mediante fijación faceta lateral. El dispositivo de fijación consiste en un canal de metal en forma de U para soportar el ratón y dos brazos ajustables de acero inoxidable que la abrazadera a cada faceta lateralmente. Esto proporciona una excelente inmovilización de la vértebra de destino. Después de la fijación espinal, la columna vertebral está ligeramente elevado para aplanar la curvatura de la columna cervical para proporcionar una mejor exposición de la médula espinal. <ol><li> Esterilizar los siguientes instrumentos quirúrgicos: 2-3 pares de pinzas, 2 pares de micro tijeras, un soporte 30 G aguja, sutura y agujas, clips de la piel y del aplicador del clip. Desinfectar el estabilizador de la columna vertebral. Anesthetize el ratón usando un cóctel intraperitoneal de ketamina / xilazina (100 mg / 10 mg / kg). Afeitar el pelo del cuello del ratón. </li><li> Después de limpiar la piel con una solución de povidona yodada y alcohol al 70%, mueva el ratón sobre la mesa de operaciones calentado con una almohadilla térmica. Cubra los ojos del animal con pomada oftálmica para prevenir la sequedad de la córnea. </li><li> Después de la inducción de la anestesia (alcanzado cuando el ratón no responde a una pizca de cola), haga una línea media incisión cervical posterior del occipucio a la grasa subcutánea-pad de la columna cervical inferior. En ampliación, realice una incisión entre los músculos trapecio en C2 y dividir los músculos semiespinoso de la cabeza. Identificación de la almohadilla de grasa submuscular facilita la disección en la capa correcta. </li><li> Extender la disección muscular línea media caudalmente a la apófisis espinosa T2, el cual sirve como un punto de referencia fiable. Cortar los músculos vinculados a la vértebra T2 y quitar la parte cartilaginosa de la T2apófisis espinosa. </li><li> Diseccionar los músculos paraespinales de la C2 a través de las láminas T2 utilizando un par de micro-tijeras. La disección del músculo comienza adyacente a las apófisis espinosas y se extiende bilateralmente a las articulaciones facetarias. Separar los músculos inmediatamente adyacentes a las apófisis espinosas y las láminas (en la capa perióstica) para minimizar el sangrado. Después de las facetas laterales están expuestos, coloque el puntero del ratón sobre el canal en forma de U de la etapa de LISA. </li><li> Coloque los brazos de acero inoxidable debajo de las caras expuestas bilateralmente. Una vez que los brazos están en su lugar, apriete los tornillos de los brazos de acero para inmovilizar la columna. Esto mantiene firme fijación de la vértebra destino y proporciona una excelente exposición. Los brazos pueden ajustarse para proporcionar la orientación horizontal exacta de la columna vertebral. </li><li> Incisión en el ligamento amarillo entre C5 y C6 exponer subyace dura. Entre el espacio interlaminar, utilizar una aguja de 30 G para crear una pequeña durotomía través de la cual micro tijeras se colocan aampliar el durotomía. La médula espinal está dispuesto a someterse a la lesión desgarro controlado. </li></ol> 2. Laceración cervical de la médula espinal Uso del dispositivo LISA <ol><li> La anchura de la ampliación de la médula espinal cervical varía en los diferentes niveles. Hacer una lesión de hemisección dorsal en C5-6 utilizando una cuchilla plana de 2,3 mm y ajustar la amplitud de la vibración para cubrir toda la anchura de la médula espinal. Las cuchillas se obtuvieron a partir de Bellas Ciencia Tools Inc. (Foster City, CA) y modificar para laceración de la médula espinal. Mantener la amplitud de la oscilación de la cuchilla en ≥ 0,5 mm, como los niveles de amplitud más bajas disminuirán la facilidad de laceración espinal. </li><li> Coloque el estabilizador de la columna vertebral y el ratón en el escenario LISA. La cuchilla está unido a la LISA con su posición controlada por micro-controladores capaces de tres rangos de movimiento. Componentes de la Lisa y sus funciones se describen en la Figura 1. </li><li> Encienda el interruptor de cuchilla vibrante sucesivamente. Bajo magnifmedicación, mueva el ratón para que la médula espinal expuesta se coloca directamente debajo de la cuchilla vibrante. </li><li> Eleve la plataforma de soporte el ratón hacia la cuchilla oscilante. La posición &quot;0&quot; se registra cuando la hoja apenas toca la vena dorsal de la médula espinal. Medir la profundidad de la laceración de la médula espinal en relación con la posición &quot;0&quot;. </li><li> Elevar la posición de fase de control micro-controlador: un giro de 360 ​​º de la perilla micro-controlador eleva la etapa en 0,25 mm. Por lo tanto, una lesión de hemisección dorsal mm 0,75 se crea girando el botón de micro-controlador 3 veces. La precisión de la lesión es de ± 0,01 mm. A medida que la hoja empieza a lacerar la médula espinal, lubricar el campo quirúrgico con irrigación salina. La profundidad de corte de la médula espinal es controlado por el micro-controlador vertical y es independiente de la orientación visual. </li><li> Una vez que se ha alcanzado la profundidad predeterminada, gire el interruptor de vibración fuera. Idealmente, la hoja oscilante se coloca en el lbrecha Esion sin evidencia de deformación del tejido. Baje la etapa de la cuchilla de corte y quitar la sangre y solución salina del campo quirúrgico con algodón Q-tips. La hemostasia se produce espontáneamente en &lt;1 min. </li><li> Suelte el botón del estabilizador de la columna vertebral. Aproximados de los músculos paravertebrales con sutura de seda 6-0 y cerrar la piel herida con clips de acero inoxidable Michel. </li></ol> 3. Cuidado de Animales <ol><li> Por vía subcutánea inyectar un total de 1-2 ml de solución salina para mantener una hidratación adecuada y coloque el puntero del ratón en la jaula de recuperación en una almohadilla eléctrica, mientras que la recuperación de la conciencia. </li><li> Proporcionar agua y alimentos blandos ad lib y administrar analgésicos durante 48 horas después de la operación. No hay necesidad para el cuidado de la vejiga después de la hemisección dorsal de la médula espinal. </li></ol>

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<ol><li> Varios autores (YPZ, XMX, CBS) tienen un interés financiero en el Louisville Impactor System, Inc. </li><li> Los autores, Yi Ping Zhang, Lisa BE Shields y Christopher B. Escudos, son empleados de Norton Healthcare, Louisville, KY. Otros autores son empleados de la Universidad de Indiana, Indianapolis, IN. </li><li> Los autores no recibieron financiación de cualquier empresa que producen reactivos o instrumentos utilizados en este artículo. </li></ol>

Estabilización vertebral antes de la laceración lesiones a la médula espinal se ha obtenido por la fijación de las apófisis espinosas. Tanto la curva de la columna cervical lordosis y la fijación de las abrazaderas de los movedizos cortos apófisis espinosas cervicales de C3 a T1 en el ratón impiden la estabilización de la columna vertebral eficaz. Además, el uso de una hoja de afeitar o micro tijeras utilizado bajo control manual provoca la deformación tisular significativa que crea variabilidad en la profundidad de la lesión 6. Esto puede conducir a la mala interpretación de los datos en particular cuando se estudia la regeneración axonal de las vías específicas. Por ejemplo, escatimado axones corticoespinales dorsales pueden ser mal interpretados como axones regenerados si el tracto corticoespinal dorsal no se secciona completamente en el momento de lesioning. Estos problemas se pueden superar mediante el uso de un dispositivo de estabilización de la columna con la fijación de las facetas a un solo nivel y lesioning precisa de la médula espinal. Además, el uso de ahcuchilla oscilante frecuencia igh produce un fuerte desgarro sin aplastar o contusing la vecina médula espinal. Este método ha sido utilizado para producir laceración lesiones de la médula espinal en ratas 9,12,14, con modificaciones posteriores para producir laceraciones de la médula espinal torácica en ratones 6. En la presente comunicación se describe el método de crear lesiones laceración cervical fiables en el ratón. En la medida en el diámetro antero-posterior de la médula espinal es &lt;2 mm en el ratón, profundidades precisas de la lesión laceración son de vital importancia en la creación de un modelo experimental fiable. Variabilidad mínima en la profundidad de la lesión va a alterar significativamente los resultados de los experimentos de evaluación de la regeneración de axones, así como estudios volumétricos y de comportamiento. La exactitud de la profundidad de la lesión utilizando este método es de ± 0,01 mm debido a que utiliza micro-controladores de alta precisión para controlar la posición de la cuchilla de corte. Este método ha reducido la falta de coherencia eninherente a otros modelos de la creación de una laceración SCI. Este método es particularmente útil en el estudio de la regeneración axonal de la larga médula espinal vías situado en la mitad dorsal de la médula espinal, tales como el tracto corticoespinal, el tracto rubroespinal y el tracto ascendente dorsal. Con este método, estos tractos de fibras pueden ser completamente fiable y seccionado. A este respecto, los errores de interpretación de los datos se reducen al mínimo, lo que mejora la fiabilidad de la presentación de informes de los estudios experimentales en SCI. En resumen, hemos descrito una nueva técnica para crear un reproducibles en el modelo in vivo de lesión espinal laceración espinal cervical en el ratón. Esta técnica se basa en la estabilización de la columna por la fijación de las facetas cervicales y laceración de la médula espinal utilizando una cuchilla oscilante. El uso de este método en un modelo de laceración dorsal de la médula espinal torácica en ratones 6, hemos demostrado una estrecha correlación entre la profundidad de laceración, histología, yrecuperación de comportamiento. Esta técnica también se ha encontrado para ser fiable por varios otros laboratorios de 2,12.

La disponibilidad de ratones modificados genéticamente es una poderosa herramienta para identificar los efectos de los genes específicos que juegan un papel en los mecanismos de SCI. Laceración SCI es un modelo importante para examinar agentes terapéuticos o moléculas que pueden proporcionar un tratamiento eficaz después de esta lesión 8. Fijación de las apófisis espinosas durante la creación de la lesión laceración en ratones es impreciso debido a la dificultad para comprender las apófisis espinosas delgadas y frágiles implicados con el mantenimiento de fijación espinal 5,11. La variabilidad en la profundidad de la laceración de sólo 0,2 mm (10% del diámetro de la médula espinal de ratón) hace que la interpretación errónea de los datos. La naturaleza y extensión de la lesión de la médula espinal laceración deben definirse con precisión 10. Para hacer frente a este desafío, hemos desarrollado una nueva técnica que consiste en la estabilización vertebral y se utiliza cuchillas fijadas al Lesiones Aparato Sistema de Louisville (LISA) para producir una laceración SCI 7,14. Esta lesión se creó utilizando una cuchilla oscilante fuerte que evita la deformación del tejido durante el proceso de laceración. La profundidad de la laceración era precisa con una exactitud de 0,01 mm mediante el uso de micro-controladores que controlan la profundidad de laceración. Cuchillas de corte están hechos a medida de las formas y anchos específicos para crear la laceración deseado contorno 9. Demostramos 1) el método de exposición columna cervical, 2) la técnica de estabilización vertebral utilizando un dispositivo de fijación bilateral de faceta, y 3) la creación de una lesión en la laceración cervical utilizando una cuchilla vibrante.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Mice vertebral stabilizer </td>
 <td>Louisville Impactor System</td>
 <td></td>
 <td>Stabilize and expose the cervical vertebra</td>
 </tr>
 <tr>
 <td>LISA vibraknife </td>
 <td>Louisville Impactor System</td>
 <td></td>
 <td>Produce the laceration injury of the cervical spinal cord</td>
 </tr>
 <tr>
 <td>Spring Scissors</td>
 <td>Fine Science Tools (USA) </td>
 <td>15013-12</td>
 <td>Skin and trapezius muscle incision</td>
 </tr>
 <tr>
 <td>Spring Scissors</td>
 <td>Fine Science Tools (USA) </td>
 <td>15023-10</td>
 <td>Separate muscles from the laminae</td>
 </tr>
 <tr>
 <td>Spring Scissors</td>
 <td>Fine Science Tools (USA) </td>
 <td>15002-08</td>
 <td>Incision of dura</td>
 </tr>
 <tr>
 <td>Graefe forceps </td>
 <td>Fine Science Tools (USA) </td>
 <td>11154-10</td>
 <td>Retract skin</td>
 </tr>
 <tr>
 <td>Dumont #7 forceps </td>
 <td>Fine Science Tools (USA) </td>
 <td>11274-20</td>
 <td>Muscle retraction (tip modified)(Fig. A)</td>
 </tr>
 <tr>
 <td>Dumont SS forceps </td>
 <td>Fine Science Tools (USA) </td>
 <td>11203-25</td>
 <td>Fixation of vertebra (tip modified )(Fig.B)</td>
 </tr>
 <tr>
 <td>30G needle </td>
 <td>Becton Dickenson</td>
 <td>305106</td>
 <td>Create a dural opening </td>
 </tr>
 <tr>
 <td>6-0 suture </td>
 <td>Ethicon </td>
 <td>8806H</td>
 <td>Close muscle and fascial layers </td>
 </tr>
 <tr>
 <td>wound clip </td>
 <td>Fine Science Tools (USA) </td>
 <td>12031-07</td>
 <td>Skin closure</td>
 </tr>
 <tr>
 <td>Tribromoethanol (Avertin)</td>
 <td>Sigma-Aldrich</td>
 <td>90710-10G</td>
 <td>Anesthetic agent</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Louisville Impactor System, Inc, 210 E. Gray St., Suite 1102, Louisville, KY 40202, (502) 629-5510, E-mail: <a href="mailto:cbshields1@gmail.com">cbshields1@gmail.com</a>
 Fine Science Tools (USA), Inc, 373-G Vintage Park Drive, Foster City, CA 94404-1139, (800) 521-2109, E-mail: <a href="mailto:info@finescience.com">info@finescience.com</a> 
 Becton Dickenson, 1 Becton Drive, Franklin Lakes, NJ USA 07417, (201) 847-6800 Ethicon, Route 22 West,
 Somerville, NJ 08876 1-877-ETHICON 
 Sigma-Aldrich Corp. 
 St. Louis, MO, USA, 63178 (314) 771-5765, E-mail: cssorders@sial.com</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Figure A is the modified Dumont #7 forceps; B is the modified Dumont SS forceps.</td>
 </tr>
 </tbody>
 </table>

controlled cervical laceration injury in mice

El uso de ratones modificados genéticamente mejora nuestra comprensión de los mecanismos moleculares que subyacen a varios trastornos neurológicos tales como una lesión de la médula espinal (SCI). Control manual Freehand utilizado para producir un modelo de laceración del SCI crea lesiones incompatibles con frecuencia asociados con un flechazo o contusión de los componentes y, por lo tanto, se ha desarrollado una nueva técnica. Nuestro modelo de cuello uterino laceración SCI ha resuelto las dificultades inherentes con el método de pulso mediante la incorporación de 1) la estabilización vertebral cervical faceta por la fijación vertebral, 2) una mayor exposición a la médula espinal, y 3) la creación de una laceración reproducible de la médula espinal usando una cuchilla oscilante con una precisión de ± 0,01 mm de profundidad sin contusión asociada. En comparación con los métodos estándar de la creación de una laceración SCI tales como el uso a mano alzada de un bisturí o unas tijeras, nuestro método ha producido una lesión consistente. Este método es útil para estudios sobre la regeneración axonal de corticospinal, rubrospinal y tractos ascendentes dorsales.

Inmovilización de la vértebra objetivo es de gran importancia en la generación de lesiones precisas de la médula espinal de ratón. Nuestro dispositivo de estabilización de la columna supera las cuestiones anatómicas de apófisis espinosas cortas y lordosis ventral del ratón de la columna cervical. Las vértebras cervicales son bien expuesta con nuestra columna vertebral estabilizador cervical (Figura 2). Nuestra columna vertebral del ratón dispositivo de estabilización es una técnica fiable para preparar la columna vertebral de los procedimientos de la médula espinal cervical. La profundidad de la lesión usando el LISA tiene una precisión de 0,01 mm 6,13. La laceración precisa no causa contusión en la interfaz de lesión / tejido (Figura 3). La precisión de las lesiones hemisección dorsal se demostró en ratones C57BL / 6 en un estudio sobre la regeneración axonal en el que una profunda laceración 0,9 mm extiende justo más allá del canal central en cada muestra confirmada por secciones patológicas de la médula espinal 1. Locomoción de todos estos animales se recuperaned después de esta lesión laceración de la médula espinal. <img alt="Figura 1" src="/files/ftp_upload/50030/50030fig1.jpg" /> Figura 1. (A) El ratón en el estabilizador de la columna vertebral colocado en la etapa de LISA. La cuchilla vibrante se dirige hacia la médula espinal a ser desgarrado. Controles Micro-controladores están ubicados debajo del escenario y están diseñados para posicionar el ratón en el lugar apropiado. El micro-controlador verticales controla la profundidad de la lesión, y el mando de inclinación controla el plano horizontal de la médula espinal para prevenir la angulación de la laceración. El interruptor de encendido y apagado controla el motor de vibración, y otro mando ajusta su amplitud. (B) A mm dorsal lesión laceración 0.75 hemisección corte bajo arcos laminares intactos. <img alt="La figura 2" src="/files/ftp_upload/50030/50030fig2.jpg" /> Figura 2. </strong&gt; (A) El estabilizador de la columna vertebral del ratón que consiste en un canal en forma de U y dos brazos y los conectores. El ratón se coloca en el canal C se utiliza para SCI cervical y en el canal T para SCI torácica. (B) La columna cervical se fija mediante la colocación de los brazos por debajo de las facetas laterales y a continuación, el bloqueo de los tornillos de mariposa. La duramadre está expuesto entre las láminas de C5-6, arilo C6-7, y C7-T1 sin la extracción del hueso. <img alt="Figura 3" src="/files/ftp_upload/50030/50030fig3.jpg" /> Figura 3. Cuatro laceraciones dorsales de la médula espinal a profundidades de 0,5, 0,8, 1,1, y 1,4 mm observados en la vista sagital (cresil-violeta y eosina) que representa el alto grado de precisión usando esta técnica.

Watch this Scientific Journal Video about Controlled Cervical Laceration Injury in Mice at JoVE.com

Controlled Cervical Laceration Injury in Mice

Una nueva técnica para crear un reproducibles<em&gt; In vivo</emSe describe&gt; modelo de cable de lesiones laceración de la médula cervical en el ratón. Esta técnica se basa en la estabilización de la columna por la fijación de las facetas cervicales y laceración de la médula espinal utilizando una cuchilla oscilante con una precisión de ± 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 ...

controlled-cervical-laceration-injury-in-mice

Research

JoVE Journal

Medicine

16.5K vistas.  Norton Healthcare. Una nueva técnica para crear un reproducibles In vivo modelo de cable de lesiones laceración de la médula cervical en el ratón. Esta técnica se basa en la estabilización de la columna por la fijación de las facetas cervicales y laceración de la médula espinal utilizando una cuchilla oscilante con una precisión de ± 0,01 mm.

Video: Controlled Cervical Laceration Injury in Mice

Pressure Controlled Ventilation to Induce Acute Lung Injury in Mice

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged mechanical ventilation, contributes to morbidity and mortality of critical illness, and studies on novel genetic or pharmacological targets are areas of intense investigation (1-3, 5, 8, 26, 30, 33-36). ALI is defined by the acute onset of the disease, which leads to non-cardiac pulmonary edema and subsequent impairment of pulmonary gas exchange (36). We have developed a murine model of ALI by using a pressure-controlled ventilation to induce ventilator-induced lung injury (2). For this purpose, C57BL/6 mice are anesthetized and a tracheotomy is performed followed by induction of ALI via mechanical ventilation. Mice are ventilated in a pressure-controlled setting with an inspiratory peak pressure of 45 mbar over 1 - 3 hours. As outcome parameters, pulmonary edema (wet-to-dry ratio), bronchoalveolar fluid albumin content, bronchoalveolar fluid and pulmonary tissue myeloperoxidase content and pulmonary gas exchange are assessed (2). Using this technique we could show that it sufficiently induces acute lung inflammation and can distinguish between different treatment groups or genotypes (1-3, 5). Therefore this technique may be helpful for researchers who pursue molecular mechanisms involved in ALI using a genetic approach in mice with gene-targeted deletion.

A murine model for ventilator induced lung injury is an important tool to study an acute lung injury in vivo. Here, we report an easy applicable in situ model for acute lung injury using high-pressure mechanical ventilation to induce acute failure of the lung.

La presión de la ventilación controlada para inducir la lesión pulmonar aguda en ratones

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged ...

Investigación

Medicina

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) ...

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 ...

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

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

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

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

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

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, ...

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. ...

Heterotopic Cervical Heart Transplantation in Mice

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies this model by avoiding challenging suture anastomoses of small vessels thereby reducing warm ischemia time. In comparison to abdominal graft implantation the cervical model is less invasive and the implanted graft is easily accessible for further follow-up examinations. Anastomoses are performed by pulling the ascending aorta of the graft over the cuff with the recipient&#8217;s common carotid artery and by pulling the main pulmonary artery over the cuff with the external jugular vein. Selection of appropriate cuff size and complete mobilization of the vessels are important for successful revascularization. Ischemia-reperfusion (I/R) injury can be minimized by perfusing the graft with a cardioplegic solution and by hypothermia. In this article, we provide technical details for a simplified and improved cuff technique, which should allow surgeons with basic microsurgical skills to perform the procedure with a high success rate.

The cuff technique shortens and facilitates the model of heterotopic cervical heart transplantation in mice by avoiding technically challenging suture anastomoses of small vessels. The technical details shown in this video paper should allow researches to establish this model in their laboratories.

Heterotópico cervical trasplante cardiaco en ratones

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies ...

A Repetitive Concussive Head Injury Model in Mice

Despite the concussion/ mild traumatic brain injury (mTBI) being the most frequent occurrence of traumatic brain injury, there is still a lack of knowledge on the injury and its effects. To develop a better understanding of concussions, animals are often used because they provide a controlled, rigorous, and efficient model. Studies have adapted traditional animal models to perform mTBI to stimulate mild injury severity by changing the injury parameters. These models have been used because they can produce morphologically similar brain injuries to the clinical condition and provide a spectrum of injury severities. However, they are limited in their ability to present the identical features of injuries in patients. Using a traditional impact system, a repetitive concussive injury (rCHI) model can induce mild to moderate human-like concussion. The injury degree can be determined by measuring the period of loss of consciousness (LOC) with a sign of a transient termination of breathing. The rCHI model is beneficial to use for its accuracy and simplicity in determining mTBI effects and potential treatments.

Concussion presents the most common type of traumatic brain injury. Therefore, a repetitive concussive animal model, which replicates the important features of an injury in patients, may provide a means to study concussion in a rigorous, controlled, and efficient manner.

Un modelo de lesión en la cabeza repetidos de conmoción en ratones

Despite the concussion/ mild traumatic brain injury (mTBI) being the most frequent occurrence of traumatic brain injury, there is still a lack of ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

Induction of Accelerated Atherosclerosis in Mice: The &quot;Wire-Injury&quot; Model

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

Inducción de la Aterosclerosis Acelerada en Ratones: El Modelo de "Lesiones por Alambre"

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...