Un movimiento versátil modelo murino de la sustancia blanca subcortical para el Estudio de la degeneración axonal y la sustancia blanca Neurobiología
Summary
Here we present methodology for the production of a focal stroke in murine white matter by local injection of an irreversible endothelial nitric oxide synthase (eNOS) inhibitor (L-Nio). Presented are two stereotactic variations, retrograde neuronal tracing, and fresh tissue labeling and dissection that expand the potential applications of this technique.
Abstract
Accidente cerebrovascular que afecta a las cuentas de la materia blanca de hasta el 25% de los accidentes cerebrovasculares presentaciones clínicas, se produce en silencio a velocidades que pueden ser 5-10 veces mayor, y contribuye de manera significativa al desarrollo de la demencia vascular. Pocos modelos de derrame cerebral focal materia blanca existen y esta falta de modelos apropiados ha dificultado la comprensión de los mecanismos neurobiológicos implicados en la respuesta lesión y reparación después de este tipo de accidente cerebrovascular. La principal limitación de otros modelos de accidente cerebrovascular subcortical es que no restrinjan de manera focal del infarto de la sustancia blanca o principalmente han sido validados en las especies no murinos. Esto limita la capacidad de aplicar la amplia variedad de herramientas de investigación murinos para estudiar la neurobiología de la carrera de la materia blanca. Aquí presentamos una metodología para la producción fiable de un derrame cerebral focal en la sustancia blanca murino mediante una inyección local de un inhibidor de eNOS irreversible. También presentamos algunas variaciones en el protocolo general entre ellos dos estereotáctica únicovariaciones, la localización neuronal retrógrada, así como el etiquetado de disección de tejido fresco y que en gran medida ampliar las posibles aplicaciones de esta técnica. Estas variaciones permiten múltiples enfoques para analizar los efectos neurobiológicos de esta forma común y poco estudiada de accidente cerebrovascular.
Introduction
Stroke affecting the subcortical white matter is a common clinical entity, accounting for up to 25% of clinical strokes annually in the US 1. Ischemic damage to white matter also occurs silently at a significantly higher rate and contributes to the development of vascular dementia 2,3. Presently, patients with this form of cerebral ischemia have few, if any treatment choices. Despite the clinical importance of this disease, few clinically relevant animal models exist 4,5.
The goal of this protocol is to produce a focal ischemic lesion within the murine white matter. This murine model of human disease allows the specific study of axonal injury response to stroke and how the cellular elements of white matter, namely oligodendrocytes and astrocytes along with axons, respond to and repair after stroke.
Previous reports have described a model of subcortical white matter stroke using endothelin-1 (ET-1) 6 that is similar to the one described here. Several key changes to the experimental protocol have been made thereby the potential uses of this model have expanded 7,8. This protocol provides a reliable and modifiable strategy to produce a focal stroke within mouse brain white matter.
The major advantages of this model are the use of a chemical endothelial nitric oxide synthase (eNOS) inhibitor N(5)-(1)-iminoethyl-L-ornithine HCl (L-Nio) 9 with no known paracrine effects on cellular elements of white matter which had been a complication of models using endothelin-1 10. In addition, the stereotactic targeting of white matter in the mouse allows the use of any variety of transgenic or knockout strains, greatly expanding the available tools to determine the effect of stroke on brain white matter. Here, two variations on this technique are described and demonstrate some of the additional variations that can be utilized to enhance the understanding of axonal and white matter damage and repair after stroke.
Protocol
Representative Results
Discussion
Un número de modelos anteriores de accidente cerebrovascular subcortical se han descrito incluyendo inyecciones focales de endotelina-1 en la cápsula interna, materia blanca subcortical y el cuerpo estriado en la rata y el ratón 12 a 14 6,15. Los modelos más recientes de pequeños accidentes cerebrovasculares focales han utilizado la inyección microembolias colesterol en la arteria carótida y la oclusión 16 photothrombotic de una sola de las arteriolas penetrantes 17. C…
Disclosures
The authors have nothing to disclose.
Acknowledgements
SN y MDD recibieron el apoyo de los NIH K08 NS083740 y el Departamento de Neurología de la UCLA. AJG reconoce el apoyo por el Dr. Miriam y la Fundación de Investigación Sheldon G. Adelson Médica y la Fundación Larry L. Hillblom. KLN agradece el apoyo del Centro de Accidentes Cerebrovasculares Asociación Americana del Corazón 14BFSC17760005 ASA-Bugher. ILL, EGS y STC fueron apoyados por el NIH R01 NS071481. JDH reconoce el apoyo de NIH K08 NS083740.
Materials
| L-N5-(1-Iminoethyl)ornithine, Dihydrochloride | Calbiochem | 400600-20MG | |
| Isoflurane | Phoenix Pharmaceutical, Inc. | NDC 57319-559-06 | |
| Capillary tubes | World Precision Instruments | 50-821-807 | |
| Picospritzer | Parker Instrumentation | Picospritzer II | |
| Stereotactic setup | Kent Scientific | KSC51725 | |
| Pipette puller | KOPF | Model 720 | |
| Stereomicroscope SZ51 | Olympus | 88-124 | |
| Fine scissors | Fine Scientific Tools | 14084-08 | |
| Forceps | Harvard Apparatus | PY2 72-8547 | |
| Curved Forceps | Harvard Apparatus | PY2 72-8598 | |
| Blunt dissection tool | Fine Scientific Tools | 10066-15 | |
| Drill | Dremel | 8220-1/28 | |
| Drill bits | Fine Scientific Tools | 19007-05 | |
| Vetbond | 3M | 1469SB | |
| Marcaine | HOSPIRA | NDC 0409-1610-50 | |
| Trimethoprim-Sulfamethaxole | STI Pharmacy | NDC 54879-007-16 | |
| Fluororuby | Fluorochrome Inc | 30mg | |
| Paraformaldehyde | Fisher | O4042-500 | |
| Sucrose | Fisher | BP220-10 | |
| Cryostat | Leica CM3050 S | 14047033518 | |
| Glass slides | Fisher | 12-544-7 | |
| Fast Green | Sigma | F7252-5G | |
| Dissection microscope | Nikon | SMZ1500 | |
| 23 gauge butterfly needle | Fisher | 14-840-35 | |
| 10X Hank's Balanced Salt Solution | Life Technologies | 14065056 | |
| 1M HEPES-KOH, pH 7.4 | Affymetrix | 16924 | |
| D-Glucose | Sigma | G8270 | |
| Sodium bicarbonate | Sigma | S5761 | |
| Cyclohexamide | Sigma | 01810 |
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