Name: photothrombosis-induced focal ischemia as a model of spinal cord injury in mice
Uploaded: 2015-07-16T15:00:00.000+00:00
Duration: 8 min 27 s
Description: Watch this Scientific Journal Video about Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice at JoVE.com

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

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

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The authors have nothing to disclose.

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

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

<table><tbody><tr><td>Rose Bengal</td><td>Sigma-Aldrich</td><td>330000</td><td>20 mg/ml in sterile saline</td></tr><tr><td>C57BL/6J</td><td>Jackson lab</td><td>664</td><td>22 - 25 g</td></tr><tr><td>Ketamine&amp;nbsp;</td><td>VEDCO</td><td>NDC-50989-996-06</td><td>100 mg/ml</td></tr><tr><td>Xylazine&amp;nbsp;</td><td>VEDCO</td><td>NDC-50989-234-11</td><td>100 mg/ml</td></tr><tr><td>Betadine solution</td><td>Purdue</td><td>NDC-67618-150-01</td><td>10% povidone iodine topical solution</td></tr><tr><td>Normal saline</td><td>Abott Laboratories</td><td>04930-04-10</td><td>For diluting RB, anaesthesia and for preventing tissue from drying</td></tr><tr><td>Artificial tears ointment&amp;nbsp;</td><td>Rugby</td><td>NDC-0536-6550-91</td><td>83% white petrolatum</td></tr><tr><td>Ethanol</td><td>Decon labs.Inc</td><td>2716</td><td>70% ethanol for disinfection</td></tr><tr><td>Metal halide lamp</td><td>EXFO, Canada</td><td>X-Cite 120 PC&amp;nbsp;</td><td>Set power at 12%</td></tr><tr><td>Spring scissors&amp;nbsp;</td><td>Fine Science Tool</td><td>15000-10</td><td>for minor dissection</td></tr><tr><td>Scissors (angled to side)</td><td>Fine Science Tool</td><td>14063-011</td><td>No. 3 handle</td></tr><tr><td>Standard scalpel</td><td>Fine Science Tool</td><td>10003-12</td><td>for removing muscle</td></tr><tr><td>Scalpel blade</td><td>Feather</td><td>2976</td><td>No. 10</td></tr><tr><td>Forceps (curved)</td><td>Fine Science Tool</td><td>11150-10</td><td>for holding tissue</td></tr><tr><td>Forceps (straight)</td><td>Fine Science Tool</td><td>11151-10</td><td>for holding tissue</td></tr><tr><td>Needle holder&amp;nbsp;</td><td>Fine Science Tool</td><td>12002-12</td><td>for suturing</td></tr><tr><td>Tissue adhesive glue</td><td>3M Vetbond</td><td>1469SB</td><td>to adhere to edges of the cut skin</td></tr><tr><td>Monofilament polypropylene&amp;nbsp;</td><td>USSC Sutures</td><td>VP-521</td><td>Size = 4-0 (for fascia)</td></tr><tr><td>Perma-hand silk</td><td>Ethicon</td><td>683G</td><td>Size = 4-0 (for skin)</td></tr><tr><td>Micro drill</td><td>Roboz Surgical Instrument Co. Inc.</td><td>RS-6300</td><td>with bone polishing drill bit</td></tr><tr><td>Laser doppler flowmeter</td><td>Moor Instruments</td><td>moorVMS-LDF1</td><td>for monitoring change in blood flow</td></tr><tr><td>Heating pad</td><td>Fine Science Tool</td><td>21052-00</td><td>to prevent hypothermia</td></tr><tr><td>Lab-Jack</td><td>Fisher scientific&amp;nbsp;</td><td>14-673-50</td><td>4 x 4 in plate to adjust the height of the animal</td></tr><tr><td>X-Y gliding stage&amp;nbsp;</td><td>Amscope</td><td>GT100</td><td>for positioning the animal under microscope&amp;nbsp;&amp;nbsp;</td></tr></tbody></table>

photothrombosis-induced focal ischemia as a model of spinal cord injury in mice

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

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

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

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

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

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

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

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

Research

JoVE Journal

Medicine

13.4K vistas.  University of Missouri. Photothrombosis is a minimally invasive and highly reproducible procedure to induce focal ischemia in the spinal cord and serves as a model of spinal cord injury in mice. 

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

Photothrombotic Ischemia: A Minimally Invasive and Reproducible Photochemical Cortical Lesion Model for Mouse Stroke Studies

The photothrombotic stroke model aims to induce an ischemic damage within a given cortical area by means of photo-activation of a previously injected light-sensitive dye. Following illumination, the dye is activated and produces singlet oxygen that damages components of endothelial cell membranes, with subsequent platelet aggregation and thrombi formation, which eventually determines the interruption of local blood flow. This approach, initially proposed by Rosenblum and El-Sabban in 1977, was later improved by Watson in 1985 in rat brain and set the basis of the current model. Also, the increased availability of transgenic mouse lines further contributed to raise the interest on the photothrombosis model. Briefly, a photosensitive dye (Rose Bengal) is injected intraperitoneally and enters the blood stream. When illuminated by a cold light source, the dye becomes activated and induces endothelial damage with platelet activation and thrombosis, resulting in local blood flow interruption. The light source can be applied on the intact skull with no need of craniotomy, which allows targeting of any cortical area of interest in a reproducible and non-invasive way. The mouse is then sutured and allowed to wake up. The evaluation of ischemic damage can be quickly accomplished by triphenyl-tetrazolium chloride or cresyl violet staining. This technique produces infarction of small size and well-delimited boundaries, which is highly advantageous for precise cell characterization or functional studies. Furthermore, it is particularly suitable for studying cellular and molecular responses underlying brain plasticity in transgenic mice.

Photothrombosis is a quick, minimally-invasive technique for inducing small and well-delimited infarction in areas of interest in highly reproducible manner. It is particularly suitable for studying cellular and molecular responses underlying brain plasticity in transgenic mice.

Isquemia photothrombotic: Un modelo de lesión cortical fotoquímico mínimamente invasiva y reproducible de Estudios Carrera Ratón

The photothrombotic stroke model aims to induce an ischemic damage within a given cortical area by means of photo-activation of a previously injected ...

Investigación

Medicina

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

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

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

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

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

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 ...

A Tissue Displacement-based Contusive Spinal Cord Injury Model in Mice

Producing a consistent and reproducible contusive spinal cord injury (SCI) is critical to minimizing behavioral and histological variabilities between experimental animals. Several contusive SCI models have been developed to produce injuries using different mechanisms. The severity of the SCI is based on the height that a given weight is dropped, the injury force, or the spinal cord displacement. In the current study, we introduce a novel mouse contusive SCI device, the Louisville Injury System Apparatus (LISA) impactor, which can create a displacement-based SCI with high injury velocity and accuracy. This system utilizes laser distance sensors combined with advanced software to produce graded and highly-reproducible injuries. We performed a contusive SCI at the 10th thoracic vertebral (T10) level in mice to demonstrate the step-by-step procedure. The model can also be applied to the cervical and lumbar spinal levels.

We introduce a tissue displacement-based contusive spinal cord injury model that can produce a consistent contusive spinal cord injury in adult mice.

Un modelo de lesión de la médula espinal contundente basado en el desplazamiento de tejidos en ratones

Producing a consistent and reproducible contusive spinal cord injury (SCI) is critical to minimizing behavioral and histological variabilities between ...

Induction of Complete Transection-Type Spinal Cord Injury in Mice

Spinal cord injury (SCI) largely leads to irreversible and permanent loss of function, most commonly as a result of trauma. Several treatment options, such as cell transplantation methods, are being researched to overcome the debilitating disabilities arising from SCI. Most pre-clinical animal trials are conducted in rodent models of SCI. While rat models of SCI have been widely used, mouse models have received less attention, even though mouse models can have significant advantages over rat models. The small size of mice equates to lower animal maintenance costs than for rats, and the availability of numerous transgenic mouse models is advantageous for many types of studies. Inducing repeatable and precise injury in the animals is the primary challenge for SCI research, which in small rodents requires high-precision surgery. The transection-type injury model has been a commonly used injury model over the last decade for transplantation-based therapeutic research, however a standardized method for inducing a complete transection-type injury in mice does not exist. We have developed a surgical protocol for inducing a complete transection type injury in C57BL/6 mice at thoracic vertebral level 10 (T10). The procedure uses a small tip drill instead of rongeurs to precisely remove the lamina, after which a thin blade with rounded cutting edge is used to induce the spinal cord transection. This method leads to reproducible transection-type injury in small rodents with minimal collateral muscle and bone damage and therefore minimizes confounding factors, specifically where behavioral functional outcomes are analyzed.

This protocol describes how to create a precise laminectomy for induction of stable transection-type spinal cord injury in the mouse model, with minimal collateral damage for spinal cord injury research.

Inducción de lesiones completas de la médula espinal de tipo transección en ratones

Spinal cord injury (SCI) largely leads to irreversible and permanent loss of function, most commonly as a result of trauma. Several treatment options, ...

Neurociencias

Modeling Stroke in Mice: Focal Cortical Lesions by Photothrombosis

Stroke is a leading cause of death and acquired adult disability in developed countries. Despite extensive investigation for novel therapeutic strategies, there remain limited therapeutic options for stroke patients. Therefore, more research is needed for pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity, and regeneration. Given the inability of in vitro models to reproduce the complexity of the brain, experimental stroke models are essential for the analysis and subsequent evaluation of novel drug targets for these mechanisms. In addition, detailed standardized models for all procedures are urgently needed to overcome the so-called replication crisis. As an effort within the ImmunoStroke research consortium, a standardized photothrombotic mouse model using an intraperitoneal injection of Rose Bengal and the illumination of the intact skull with a 561 nm laser is described. This model allows the performance of stroke in mice with allocation to any cortical region of the brain without invasive surgery; thus, enabling the study of stroke in various areas of the brain. In this video, the surgical methods of stroke induction in the photothrombotic model along with&#160;histological analysis are demonstrated.

Described here is the photothrombotic stroke model, where a stroke is produced through the intact skull by inducing permanent microvascular occlusion using laser illumination after administration of a photosensitive dye.

Modelado de accidente cerebrovascular en ratones: lesiones corticales focales por fototrombosis

Stroke is a leading cause of death and acquired adult disability in developed countries. Despite extensive investigation for novel therapeutic strategies, ...

Establishing a Mouse Contusion Spinal Cord Injury Model Based on a Minimally Invasive Technique

Using minimally invasive methods to model spinal cord injury (SCI) can minimize behavioral and histological differences between experimental animals, thereby improving the reproducibility of the experiments.
These methods need two requirements to be fulfilled: clarity of the surgical anatomical pathway and simplicity and convenience of the laboratory device. Crucially for the operator, a clear anatomical pathway provides minimally invasive exposure, which avoids additional damage to the experimental animal during the surgical procedures and allows the animal to maintain a consistent and stable anatomical morphology during the experiment.
In this study, the use of a novel integrated platform called the SCI coaxial platform for spinal cord injury in small animals to expose the T9 level spinal cord in a minimally invasive way and stabilize and immobilize the vertebra of mice using a vertebral stabilizer is researched, and, finally, a coaxial gravity impactor is used to contuse the spinal cord of mice to approach different degrees of T9 spinal cord injury. Finally, histological results are provided as a reference for the readers.

Minimally invasive techniques and a simple laboratory device improve the reproducibility of the spinal cord injury model by reducing operative damage to the experimental animals and allowing anatomical morphology maintenance. The method is worthwhile because the reliable results and reproducible procedure facilitate investigations of the mechanisms of disease reparation.

Establecimiento de un modelo de lesión de la médula espinal por contusión en ratones basado en una técnica mínimamente invasiva

Using minimally invasive methods to model spinal cord injury (SCI) can minimize behavioral and histological differences between experimental animals, ...

Establishment of Central Cord Syndrome Model in C57BL/6J Mouse

Animal models of central cord syndrome (CCS) could substantially benefit preclinical research. Identifiable anatomical pathways can give minimally invasive exposure approaches and reduce extra injury to experimental animals during operation, enabling the maintenance of consistent and stable anatomical morphology during experiments to minimize behavioral and histological differences between individuals to improve the reproducibility of experiments. In this study, the C6 level spinal cord was exposed using a spinal cord injury coaxial platform (SCICP) and combination with a minimally invasive technique. With the assistance of a vertebral stabilizator, we fixed the vertebrae and compressed the spinal cords of C57BL/6J mice with 5 g/mm2 and 10 g/mm2 weights with SCICP to induce different degrees of C6 spinal cord injury. In line with the previous description of CCS, the results reveal that the lesion in this model is concentrated in the gray matter around the central cord, enabling further research into CCS. Finally, histological results are provided as a reference for the readers.

The present protocol simulating central cord syndrome (CCS) in mice has improved repeatability and minimized operation damage to the experimental animals, avoiding disrupting the anatomical structure excessively. The strategy in this study is advantageous because it allows for research into injury mechanisms by producing consistent results.

Establecimiento del modelo de síndrome de cordón central en ratón C57BL/6J

Animal models of central cord syndrome (CCS) could substantially benefit preclinical research. Identifiable anatomical pathways can give minimally ...

Modeling Photothrombotic Stroke in Mouse Model: A Laser Illumination Technique Through Mouse Skull Following Photosensitive Dye Administration to Induce Photothrombosis

Source: Llovera, G. et al. <a href="https://www.jove.com/v/62536">Modeling Stroke in Mice: Focal Cortical Lesions by Photothrombosis.</a> J. Vis. Exp. (2021)
This video demonstrates the procedure to generate photothrombotic stroke in a mouse model by laser illumination of an intact skull following administration of photosensitive dye.

Modelado de accidente cerebrovascular fototrombótico en modelo de ratón: una técnica de iluminación láser a través del cráneo de ratón después de la administración de un tinte fotosensible para inducir fototrombosis

Source: Llovera, G. et al. Modeling Stroke in Mice: Focal Cortical Lesions by Photothrombosis. J. Vis. Exp. (2021)
This video demonstrates the procedure ...

JoVE Encyclopedia of Experiments

Biología

Rodent Models

Adult

Inducing a Spinal Cord Contusion in a Mouse Model Using a Spinal Cord Injury Coaxial Platform

<a href='https://app.jove.com/v/64538/establishing-mouse-contusion-spinal-cord-injury-model-based-on'>Source: Elzat, E. Y. Y., et al.,&#160;Establishing a Mouse Contusion Spinal Cord Injury Model Based on a Minimally Invasive Technique. J. Vis. Exp.&#160;(2022).</a>This video demonstrates the method for inducing a contusion injury at the T9 level spinal cord in a mouse model, using a spinal cord injury coaxial platform. A weight is dropped on the T9 level spinal cord that creates a contusion that immediately causes tissue discoloration and swelling.